The insect brain is traditionally subdivided into the trito-, deuto- and protocerebrum. However, both the neuromeric status and the course of the borders between these regions are unclear. The Drosophila embryonic brain develops from the procephalic neurogenic region of the ectoderm, which gives rise to a bilaterally symmetrical array of about 100 neuronal precursor cells, called neuroblasts. Based on a detailed description of the spatiotemporal development of the entire population of embryonic brain neuroblasts, a comprehensive analysis was carried out of the expression of segment polarity genes (engrailed, wingless, hedgehog, gooseberry distal, mirror) and DV patterning genes (muscle segment homeobox, intermediate neuroblast defective, ventral nervous system defective) in the procephalic neuroectoderm and the neuroblast layer (until stage 11, when all neuroblasts are formed). The data provide new insight into the segmental organization of the procephalic neuroectodem and evolving brain. The expression patterns allow the drawing of clear demarcations between trito-, deuto- and protocerebrum at the level of identified neuroblasts. Furthermore, evidence is provided indicating that the protocerebrum (most anterior part of the brain) is composed of two neuromeres that belong to the ocular and labral segment, respectively. These protocerebral neuromeres are much more derived compared with the trito- and deuto-cerebrum. The labral neuromere is confined to the posterior segmental compartment. Finally, similarities in the expression of DV patterning genes between the Drosophila and vertebrate brains are discussed (Urbach, 2003a).

In the trunk neuroectoderm, segment-polarity genes are expressed in
stereotypical segmental stripes, and in NBs that delaminate from these
domains, subdividing each neuromere along the AP axis. In the
pregnathal head region the expression domains of segment polarity genes are less obvious, but analysis of engrailed and wingless expression in the head peripheral ectoderm, and of PNS
mutant phenotypes, support the existence of four pregnathal segments in
Drosophila: the intercalary, antennal, ocular and labral segments (from posterior to anterior). However, the identity and organization of brain structures
deriving from these segments is still obscure. In order to obtain evidence concerning the number and extent of the brain neuromeres, and to map the position of their boundaries, the expression of segment polarity genes, including wingless, hedgehog, gooseberry-distal, engrailed, invected and mirror, was analyzed. The spatiotemporal pattern of their expression was traced in the neuroectoderm and in the NB-layer until stage 11,
when all brain NBs are formed. The data
show that segmental expression is retained for most of the investigated segment polarity genes in both the developing head ectoderm (procephalon) and brain NBs, providing landmarks for the definition of segmental domains within the developing brain NB pattern (Urbach, 2003a).

engrailed (en) expression domains in the trunk define the posterior segmental compartments, from which NBs of row 6 and 7 and NB1-2 derive. In the pregnathal head en expression was found as follows: from late stage 8 in the posterior ectoderm of the antennal segment (en antennal stripe; en as) from which four deutocerebral NBs (Dv8, Dd5, Dd9, Dd13) delaminate; from stage 9 in a small ectodermal domain in the posterior part of the ocular segment, the en head spot (en hs), from which two protocerebral NBs (Ppd5, Ppd8) evolve; and from stage 10 in an ectodermal stripe in the posterior intercalary segment (en intercalary stripe; en is), which gives rise to three to four tritocerebral NBs (Tv4, Tv5, Td3, Td5). Furthermore, from stage 11 onwards, En is weakly detected in the anteriormost ectoderm of the procephalon corresponding to the region of the 'anterior dorsal hemispheres' (en dh). About 10 weakly En-positive NBs were identified that delaminate from the en dh. Thus all four pregnathal head segments contribute to the early embryonic brain. The spatial distribution of the En-positive NBs closely corresponds to the en domains of their origin in the ectoderm. This suggests they demarcate the posterior borders of the respective brain neuromeres (Urbach, 2003a).

In the trunk, hedgehog (hh) matches en expression. This is also the case for the intercalary segment in the pregnathal head ectoderm. By contrast, the En-positive antennal stripe and head spot are only subfractions of the large hh-lacZ domain, which, between stages 9 and 10, encompasses the antennal segment and the posterior part of the ocular segment. All NBs delaminating from this domain express hh-lacZ. From stage 10 onwards, en expressing NBs maintain a strong hh-lacZ signal, whereas hh-lacZ subsequently diminishes in the neuroectoderm and in NBs between the en antennal stripe and head spot. Additionally, hh-lacZ-expressing NBs positioned dorsally to the en/hh-lacZ-co-expressing Ppd5 and Ppd8 (both NBs demarcating part of the posterior border of the ocular neuromere), appear to prolong the boundary between the deuto- and proto-cerebrum in the dorsal direction (Urbach, 2003a).

From late stage 8 onwards, Wingless (Wg) protein is expressed in a neuroectodermal domain spanning a broad area of the ocular and the anterior antennal segment (and in the invaginating foregut). This becomes clearer in En/Wg double labelling at stage 9, revealing that the en hs is localized within this Wg domain. At that stage, Wg is already detectable in about 4-5 protocerebral NBs (Pcd6, Pcd15, Pcd7, Ppd3), derived from the region with strongest Wg expression (which later corresponds to the wg head blob). Furthermore, Wg is faintly expressed in the deutocerebral Dd7 emerging from the antennal part of the Wg domain, which corresponds to the later wg antennal stripe. By stage 10, when the wg head blob is clearly distinguishable from the wg antennal stripe, about 10-12 Wg-positive NBs have emerged from this domain. In addition, a small, spot-like wg domain was found in the intercalary segment from which a single NB (Td4) delaminates. Thus, all three wg domains, the intercalary, antennal and ocular (head blob), contribute to the anlage of the brain. From late stage 9 an additional wg domain is visible in the ectodermal anlage of the clypeolabrum, which is the wg counterpart to the En/Inv-positive region in the 'dorsal hemispheres'. Upon double labelling for either asense or deadpan (both are general markers for neural precursor cells) and wg, in embryos between stage 9 and 11 no NB emerging from the wg labral spot could be detected. By stage 11 the number of wg expressing NBs originating from the ocular head blob has increased to about 16-20, which is more than 25% of the total number of identified protocerebral NBs. Three Wg-positive NBs are identified in the deutocerebrum and one in the tritocerebrum (Urbach, 2003a).

The gooseberry (gsb) locus encodes two closely related proteins, Gsb-distal (Gsb-d) and Gsb-proximal, which are both expressed in the developing ventral nerve cord. Gsb-d is segmentally expressed at high levels in all row 5 and 6 NBs, as well as in a median row 7 NB (NB 7-1). The expression of gsb-d was analyzed during early neurogenesis in the head region; segmental expression of Gsb-d was found to be conserved in parts of the pregnathal head ectoderm and deriving NBs. Gsb-d/En double labelling shows that the gsb-d intercalary and antennal stripes are expressed anterior to the corresponding en stripes, and are partly overlapping with the en stripes. Consequently, NBs from the posterior part of the gsb-d stripe in the tritocerebrum and deutocerebrum co-express en (Td3, Dd5), and those from the anterior part co-express wg (Td4, Dd1 and Dd7; as seen in Gsb-d/Wg double labelling), resembling the situation in the ventral nerve cord. However, Dd8 and all Wg-positive protocerebral NBs do not co-express Gsb-d (except for Ppd3 which, like Ppd10, transiently expresses gsb-d during stage 10. Gsb-d can also be detected at a low level in ganglion mother cells of the respective NBs, but fades away in NBs and their progeny during germ band retraction. Expression of the protein in the brain is completely downregulated at stage 13 (Urbach, 2003a).

In the trunk, mirror (mirr)-lacZ is expressed in segmental ectodermal stripes giving rise to mirr-lacZ-positive NBs of row 2 and several NBs that flank row 2 at stage 11. The pattern of mirr-lacZ expression in the procephalic neuroectoderm and brain NBs differs significantly from the trunk. No evidence is found of a segmental arrangement of mirr-lacZ expression in the procephalon. Interestingly, regarding the DV axis, mirr-lacZ is mainly limited to the ventral part of the procephalic neuroectodermal region (pNR) and corresponding NBs (as confirmed by mirr-lacZ/Vnd double staining, although there is a faint dorsal mirr-lacZ expression, in the region of the later invaginating optic lobe anlage, and is, at stage 9/10, roughly complementary to en, wg and gsb-d expression, the domains of which are mainly confined to intermediate and dorsal regions of the pNR. At stage 11, expression extends towards the dorsal part of the antennal neuroectoderm and is observed in all NBs of the ventral deutocerebrum, as well as in two tritocerebral (Tv5, Td8) and four ventral, protocerebral NBs (Pad1, Pcv1, Pcv2, Pcv3). Although expression is also found in the clypeolabrum, no mirr-lacZ-positive labral NBs were identified (Urbach, 2003a).

In addition to the segment polarity genes, the dorsoventral patterning genes ventral nervous system defective (vnd), intermediate neuroblast defective (ind) and muscle segment homeobox (msh) have been shown to confer positional information to the truncal neuroectoderm, which also contributes to the specification of NBs. For the head and brain, a detailed analysis of the expression of these genes has not yet been undertaken. In order to elucidate their putative role in patterning the head and brain, the expression of vnd, ind and msh was analyzed in the procephalic ectoderm and NBs in the early embryo (until stage 11). Although the data are consistent with their role in dorsoventral patterning being principally conserved in the procephalon, significant differences are found in their patterns of expression compared with the trunk (Urbach, 2003a).

At the blastodermal stage, Ventral nervous system defective protein (Vnd) is expressed in bilateral longitudinal stripes corresponding to the most ventral neuroectodermal column, and is by stage 11 detected in all ventral and two intermediate NBs of the ventral nerve cord. Interestingly, the latter co-express en and are located in the posterior compartment of each truncal neuromere. At gastrulation the ventral longitudinal vnd domain reaches anteriorly across the cephalic furrow into the procephalic neuroectoderm. By stage 9, vnd maps in the ventral neuroectoderm of the prospective intercalary, antennal and ocular segment and is observed in ventral NBs of the antennal (Dv2, Dv3, Dv6) and ocular neuromere (Pcv1, Pcv3, Pcv6, Ppv2). It appears as if the dorsal part of the Vnd-positive antennal neuroectoderm partly co-expresses ind at that stage, but the NB Dd1, which emerges from this ectodermal region expresses only ind and not vnd. This is possibly due to the transient expression of vnd in most parts of both the ventral antennal ectoderm and corresponding NBs: by stage 10 Vnd is detected in the ventral Dv2, Dv4 and Dd5, but is already downregulated in Dv3 and Dv6, and by stage 11 it is confined to Dd5 and the new Dv8. As a consequence of the downregulation of vnd, some ventral deutocerebral NBs, which delaminate between stage 9 and 11 from this domain were not observed to express vnd (e.g. Dv1, Dv5, Dv7). By stage 11 Vnd is seen in four tritocerebral NBs (Tv2, Tv3, Tv4, Tv5), in two deutocerebral NBs (Dd5, Dv8), and in a cluster of about 13 protocerebral NBs. Interestingly, vnd expression expands along the posterior border of the en intercalary stripe (en is), and is also significantly extended dorsally into the en antennal stripe; the NBs delaminating from there. The fact that vnd and en are co-expressed in Tv5 and in Dd5, Dv8 is in agreement with findings in the ventral nerve cord, where these genes are co-expressed in two intermediate NBs. This indicates that vnd demarcates the ventral part of the posterior border in trunk as well as in brain neuromeres. Furthermore, the posterior border of the ocular vnd domain (including the NBs Pcv1, Pcv2, Pcv3, Ppv1, Ppv2, Ppv3) abuts dorsally the En-positive NBs Ppd5 and Ppd8 (deriving from the en head spot), supporting the view that these NBs demarcate the posterior border of the ocular neuromere (Urbach, 2003a).

intermediate neuroblast defective (ind) is expressed in the blastoderm in a bilateral longitudinal column (intermediate column neuroectoderm) just dorsal to the vnd domains. In the trunk, at stage 9 (when ind mRNA is no longer present in the neuroectoderm), it is expressed in all intermediate NBs and finally, at stage 11, it is confined to the NB 6-2. In the head, at stage 9, ind is detected in an intermediate longitudinal ectodermal domain in the intercalary segment, and weakly in an intermediate ectodermal patch in the antennal segment as well as in the deutocerebral NB Dd1 which develops from this patch. At the same stage, a further signal is observed in a dorsal ectodermal patch of the ocular region. The ectodermal ind patches in the intercalary, antennal and ocular segments are both separate from each other and from the ind domain in the trunk. Interestingly, ind mRNA is significantly present longer in the ectoderm of the intercalary and mandibular segment, when compared with the antennal segment and the trunk ectoderm. This presumably mirrors the delayed onset of neurogenesis in both segments. Until stage 10, five NBs derive from the three ind patches: Td1, Td2, Td3, from the intercalary, Dd1 from the antennal and Ppd13 from the ocular ind patch. Subsequently, the ocular ind patch enlarges but never reaches the ocular vnd domain, and by stage 11 about four additional Ind expressing NBs (Pcd7, Pcd13, Ppd6, Ppd9) are identifiable (Urbach, 2003a).

muscle segment homeobox (msh) expression is first detected at the blastoderm stage in discontinuous patches in the dorsolateral part of the neuroectoderm that later extend and form a bilateral longitudinal stripe; this domain gives rise to the lateral NBs of the ventral nerve cord. At stage 7 msh expression is detected anterior to the cephalic furrow, which expands until stage 9 to cover, as a broad domain, the dorsal ectoderm of the intercalary and the antennal segment. As evidenced by Msh/Inv double labelling during stage 9 and stage 11, the anterior border of the msh domain coincides with the posterior border of the en hs. This suggests that msh expression in the pregnathal region is restricted to the intercalary and antennal segments, and matches the border between the antennal and ocular segment. This is further supported by Msh/hh-lacZ double labelling in stage 11 embryos, using hh as a marker for the posterior border of the ocular segment. All identified brain NBs delaminating from the dorsal intercalary and antennal neuroectoderm express msh. This suggests that during early neurogenesis, msh controls dorsal identities of the procephalic neuroectoderm and brain NBs, as was shown for the ventral nerve cord. In the ventral nerve cord, most glial precursor cells (glioblasts and neuroglioblasts) derive from the dorsal neuroectoderm, and express msh. In the intercalary segment of the early brain, two glial precursors (Td4 and Td7) were identified. Interestingly, both precursors are also located dorsally and express msh. At least until stage 11 no msh expression is found in the preantennal segments (Urbach, 2003a).

Comparing the expression of DV patterning genes in the trunk and
procephalic region, the following significant differences were observed (Urbach, 2003a):

Whereas msh is expressed in all segments of the trunk, it is
not expressed in the preantennal head ectoderm.

ind is expressed as a continuous stripe in the trunk, but forms
three segmental patches in the procephalon. ind expression in the
antennal segment appears to overlap with transient vnd expression, yet
this ectodermal region gives rise to Dd1 which expresses ind but not vnd.

The msh and vnd domains partially share a common border
in the intercalary and antennal segment by stage 9, and furthermore show
a partial overlap in the antennal ectoderm by stage 10/11. The En-positive
Dd5 co-expresses msh and vnd, whereas
co-expression of msh and vnd was not observed in NBs of the
ventral nerve cord.

In the ocular segment, the ind domain is separated from the
vnd domain, whereas in the trunk neuroectoderm these domains are
adjacent to one another.

vnd expression is dynamic and from stage 9 onwards is
downregulated in parts of the antennal neuroectoderm and deutocerebral NBs.

More than half of the total number of identified brain NBs do not express
any of these DV patterning genes. Most of these NBs derive from the
preantennal segments.

This implies that other, still unknown factors might be involved in the DV
patterning of the anterior head neuroectoderm and protocerebrum (Urbach, 2003a).

With regard to the expression of the segment polarity genes en, hh,
wg and gsb-d, as well as the DV patterning genes msh
and vnd, it is proposed that the procephalic (pregnathal) neuroectoderm gives rise to four brain neuromeres: the tritocerebrum, the deutocerebrum, the ocular and the labral neuromere (from posterior to anterior). These tightly fused neuromeres form a supraoesophageal brain hemisphere on either side. The ocular and labral neuromeres represent the most prominent part of the brain which is traditionally referred to as the protocerebrum (Urbach, 2003a).

The detailed analysis of the dynamic expression of these genes in the procephalic neuroectoderm and in the identified brain NBs allows mapping of the boundaries of the brain neuromeres. The posterior border of the tritocerebrum is clearly represented by the en- and hh co-expressing NBs Tv4, Tv5, Td3, Td5. In the antennal and preantennal neuroectoderm the expression of en, hh, wg and gsb-d is largely restricted to intermediate and dorsal regions, and NBs deriving from there. Thus, regarding segment polarity genes, a clear demarcation of the antennal and preantennal neuromeres is only possible for the intermediate and dorsal, but not for the ventral domains. vnd is observed to be co-expressed with en in some tritocerebral (Tv5) and deutocerebral NBs (Dv8 and Dd5), located at intermediate DV positions. This is consistent with observations in the trunk, where vnd expression is dorsally expanded into each en domain in the neuroectoderm, as well as at the level of NBs. It is therefore suggested that the (transiently) vnd expressing NBs Dv2 and Dv4, which follow Dd5 and Dv8 ventrally, demarcate the ventral part of the posterior border of the deutocerebrum. The intermediate part of this border is defined by the en/hh/vnd-co-expressing Dv8, Dd5, and the dorsal part by the en- and hh-co-expressing Dd9 and Dd13. For the posterior border of the ocular neuromere, the following is proposed. Under the assumption that vnd expression also marks the posterior compartment in this neuromere, the vnd expressing NBs Pcv1, Pcv2, Pcv3, Ppv1, Ppv2 and Ppv3 would demarcate the ventral part of this border. The intermediate part is defined by the en/hh-co-expressing Ppd5 and Ppd8, and the dorsal part by the Hh-lacZ-positive NBs Ppd10, Ppd11, Ppd15 and Ppd16. Interestingly, the anterior border of the msh domain abuts exactly on the posterior ocular segmental border, indicating that msh expression is confined to the trito- and deuto-cerebrum. inv expression is observed in about 10 NBs deriving from the most anterior part of the protocerebral anlage, a region that corresponds to the En-positive 'dorsal hemispheres' (en dh). It is suggested that these NBs represent the neural correlate of the labral segment. This fourth brain neuromere seems to be of rudimentary character since it is confined to the posterior segmental compartment (considering that en/inv is normally expressed in the posterior compartment), and no NBs anterior to en dh are found. Thus, the wg domain in the clypeolabral ectoderm, which is located immediately anterior to the en dh does not give rise to brain NBs. The existence of four brain neuromeres, in the spatial orientation shown, is furthermore substantiated by the segmental expression of other genes like gsb-d, sloppy paired 1 and ladybird (Urbach, 2003a).

Thus, these data clearly support the view that the pregnathal head consists of four segments (antennal, intercalary, ocular and labral). Furthermore, it was possible to attribute to each of the four pregnathal head segments a corresponding neuromere. All segment polarity genes are segmentally expressed in the pNR as well as in brain NBs, except mirr, the segmental expression of which is not overt. wg and gsb-d are partly overlapping, and are expressed anterior to the respective en domains, which are colocalized with hh. The expression of these genes is either mainly confined to intermediate and dorsal regions of the antennal and ocular segment (in case of en, wg and gsb-d) or is at least stronger (hh) in these parts of the pNR. Consequently, with regard to segment polarity genes there is a clear segmental demarcation, which is limited to intermediate and dorsal parts of the respective neuromeres, but it remains unclear in their ventral parts (except in the tritocerebrum). Surprisingly, the DV patterning genes vnd and msh endorse a separation of brain neuromeres in AP axis. vnd expression demarcates the ventral part of the posterior border of the tritocerebrum, deutocerebrum and ocular neuromere, and msh the dorsal anterior border of the deutocerebrum. Thus, based on the expression of segment polarity genes (en/inv, hh) and DV patterning genes (vnd, msh) a reconstruction is provided of segmental boundaries in the developing brain on the level of identified cells (Urbach, 2003a).

The segmental organization of the anterior head, in particular the origin of the labrum, the existence of a corresponding segment and its position at the anterior pole, are central issues of a long-lasting debate concerning head segmentation. Consequently, the segmental origin of the protocerebrum, the largest and most anterior portion of the brain, has been a matter of debate and there is disagreement about whether it can be assigned to the labral and/or the ocular segment (equivalent to the acron). en expression in the en dh has been attributed to the labral segment, the existence of which is further substantiated by PNS phenotypes in head gap mutants. About 10 NBs have been identified that derive from this domain and weakly express en. Immediately anterior to the en dh, within the clypeolabral ectoderm, the genes wg, gsb-d, lbe and slp1 are found to be expressed, but these domains do not contribute to the brain. The spatial pattern of expression of these genes confirms the following: the anteroposterior orientation of a labral segment, and a parasegmental character of the border between the en dh and the labral wg domain, supporting the view that the en dh is the en-expressing part of the labral segment. It is therefore concluded that the protocerebrum consists of two neuromeres, a large ocular neuromere (comprising more than 60 NBs) and a smaller labral neuromere (comprising about 10 NBs). Since en expression delimits the posterior compartment of each segment, the labral neuromere appears to be confined to the posterior compartment (Urbach, 2003a).

The protocerebrum develops prominent neuropile structures such as the central complex and the mushroom bodies. On comparative morphological grounds, the protocerebrum in arthropods has been subdivided into the archicerebrum and prosocerebrum. Accordingly, the archicerebrum, which bears the optic lobes and mushroom bodies, belongs to the acron (or ocular segment), and the prosocerebrum, which comprises the remainder of the protocerebrum (including the central complex and the neurosecretory cells of the pars intercerebralis) belongs to the labral segment. The progenitor cells of the mushroom bodies are part of the ocular neuromere, supporting the view that the mushroom bodies are indeed neuropil structures of the ocular segment or archicerebrum. Consequently, the identified labral NBs would be progenitors of neurons of the pars intercerebralis. This appears likely because during further embryogenesis the en dh becomes displaced in a brain region corresponding to the pars intercerebralis of postembryonic stages. In Drosophila, little is known about the embryonic origin of the central complex. In the grasshopper, NBs in the pars intercerebralis contribute neurons to the central complex. Taking into consideration that the identified labral NBs presumably represent the progenitors of cells of the pars intercerebralis and that the fundamental 'bauplan' of the brain is believed to be conserved among insects, it is suggested that in Drosophila, progeny cells of labral NBs participate in the formation of the central complex (Urbach, 2003a).

In the trunk, the neuroectoderm and NB pattern of each hemisegment is subdivided by the activity of segment polarity genes into transverse rows and by the activity of DV patterning genes into longitudinal columns. This orthogonal expression of segment polarity and DV patterning genes is principally conserved in the posterior part of the pregnathal head neuroectoderm and corresponding regions of the early brain, but becomes obscure towards anterior sites. The intercalary neuroectoderm and neuromere are subdivided by en, hh, wg and gsb-d expression into transverse-like rows and by msh, ind and vnd into longitudinal columns. Analysis of other genes that are segmentally expressed in the trunk CNS (e.g., slp1 and lbe) provides further support for the notion that the tritocerebrum behaves like a reduced trunk neuromere. Similarly, this orthogonal pattern of segment polarity and DV patterning gene expression appears to be essentially retained in the antennal neuroectoderm and deutocerebrum. However, it appears less conserved compared with the tritocerebrum because en, wg and gsb-d (and slp1) expression is confined to intermediate/dorsal sites, ind is restricted to one NB and vnd is only transiently expressed. The orthogonal expression pattern of both gene groups is to a minor extent, if at all, conserved in the posterior half of the ocular neuromere. Owing to the lack of msh expression, a dorsoventral polarity is less obvious and most ocular NBs do not express any DV patterning gene. Finally, conservation of this pattern is not evident in the labral segment. Although some segment polarity genes are expressed in the labral ectoderm, expression of DV patterning genes is missing (except for the two vnd-positive NBs, Pav1 and Pcv4, at the border to the ocular neuromere) (Urbach, 2003a).

In this context, it is interesting to note that the head has been claimed to be composed of two distinct domains, an anterior terminal domain and a segmented region. Both domains require high levels of Bicoid protein as an anterior determinant, but the anterior terminal domain, which encompasses the labral segment and the acron (which is equivalent to the ocular segment) is primarily specified by a signalling pathway mediated by the receptor tyrosine kinase Torso. Zygotic target genes which become activated by this signalling pathway are the gap genes hkb and tll. For tll, it has been shown that (part of) its anterior, blastodermal expression is necessary for the development of the protocerebrum, which is missing in tll mutants. tll represses hb and ftz and may thus function in the head as an 'anti-segmentation' gene. tll expression, which covers the ocular and labral neuroectoderm (the latter coincides with the region of the en dh) and emerging NBs, closely corresponds to that part of the early brain where segmental features are largely obscure. A coordinated, orthogonal expression of segment polarity and DV patterning genes within the ocular and labral neuroectoderm is not obvious, and the existence of putative serially homologous NBs in those regions of the brain is less evident. This implies that tll might be a component crucial for the suppression of segmental characteristics in the ocular and labral neuromere. Furthermore, crossregulatory interactions among the segment polarity genes in the pregnathal head differ from those in the trunk and are unique for each pregnathal segment (Urbach, 2003a).

For a part of the segmented head (mandibular, intercalary and antennal) it has been proposed that a combinatorial expression of the cephalic gap genes otd, ems and buttonhead mediates metamerization by acting directly on segment polarity genes, thereby omitting the intermediate function of pair rule genes. Recent data indicate that, in the segmental patterning of this head region, other (intermediate) regulators are involved. One of these is collier, which is already expressed in the blastoderm and is required for the formation of the intercalary segment. It is controlled by the combined activity of ems and buttonhead, and the pair rule gene even skipped, thus integrating inputs from both the head and trunk segmentation system. Such factors might help to explain that trunk specific segmental characteristics are more conserved in the intercalary and antennal neuroectoderm and NBs, when compared to the ocular and labral neuroectoderm and NBs (Urbach, 2003a).

In Drosophila the DV patterning genes subdivide the trunk neuroectoderm into longitudinal columns; vnd is required for the specification of the ventral neuroectodermal column and NBs; ind and msh have analogous functions in the intermediate and dorsal neuroectodermal columns and NBs, respectively. Remarkably, homologous genes are found to be expressed in the vertebrate neural plate and subsequently in the neural tube. In the neural tube the order of expression along the DV axis is analogous to that of Drosophila: like vnd, the vertebrate homologs of the Nkx family are expressed in the ventral region; the ind homologs, Gsh-1/2, are expressed in the intermediate region; and the msh homologs, Msx-1/2/3, are expressed in the dorsal region of the neural tube (Urbach, 2003a).

These DV patterning genes are expressed in the procephalic neuroectoderm and developing brain. Furthermore, it has been observed that in the anterior, the extent of expression is specific for each gene: msh is confined to more posterior regions, and vnd expression extends into anterior regions of the brain. Moreover, the expression border of msh and vnd coincide with neuromeric borders. A comparison of the anteroposterior sequence of DV patterning gene expression in the early brain of Drosophila, with that published for the early mouse brain, reveals striking similarities. Msx3, which presumably represents the ancestral msh/Msx gene, becomes restricted to the dorsal neural tube during later development (in contrast to Msx1/2). The anterior border of the Msx3 domain is positioned within the rostral region of the dorsal rhombencephalon, thus showing the shortest rostral extension of all vertebrate DV patterning genes. This displays analogy to msh, the expression domain of which coincides with the anterior border of the dorsal deutocerebrum, thus representing the shortest anterior extension of DV patterning genes in Drosophila. Mouse Nkx2.2 extends ventrally into the most rostral areas of the forebrain. vnd is expressed ventrally in anterior parts of the ocular and labral protocerebrum. Thus, the expression of the respective homologs in both species displays the most anterior extension among DV patterning genes. Moreover, Nkx2.2 expression in the mouse forebrain suggests that Nkx2.2 may be involved in specifying diencephalic neuromeric boundaries. Similarly, in Drosophila, dorsal expansions of the vnd domain appear to correspond to the tritocerebral and deutocerebral neuromeric boundaries (Urbach, 2003a).

Furthermore, Drosophila ind and its mouse homologue Gsh1 show similarities in their expression in the early brain. In the posterior parts of the Drosophila brain, ind is expressed in intermediate positions between vnd and msh. Likewise, in the posterior part of the mouse brain, Gsh1 appears to be expressed in intermediate positions, dorsally to Nkx2.2, and in the hindbrain ventrally to Msx3. Gsh1 has been shown to be expressed in discrete domains within the mouse hindbrain, midbrain (mesencephalon) and the most anterior domain in the posterior forebrain (diencephalon). Correspondingly, in Drosophila ind expression in restricted domains within the gnathocerebrum, the tritocerebrum, deutocerebrum and ocular part of the protocerebrum, demonstrating that the anteriormost extension of ind (and Gsh1) expression lies between that of msh and vnd (Urbach, 2003a).

Taken together, considering these similarities, it is suggested that in the Drosophila and vertebrate early brain the expression of DV patterning genes is to some extent conserved, both along the DV axis (as suggested for the truncal parts of the Drosophila and mouse CNS) and along the AP axis. Furthermore, in Drosophila large parts of the anterodorsal procephalic neuroectoderm and NBs (more than 50% of all identified brain NBs) lack DV patterning gene expression. Likewise, in the vertebrate neural tube, gaps between the expression domains of DV patterning genes have been described, raising the possibility that other genes might fill in these gaps. How DV fate is specified in the anterior and dorsal part of the Drosophila procephalic neuroectoderm, and if other genes are involved, remains to be clarified (Urbach, 2003a).

Molecular markers for identified neuroblasts in the developing brain of Drosophila

The Drosophila brain develops from the procephalic neurogenic region of the ectoderm. About 100 neural precursor cells (neuroblasts) delaminate from this region on either side in a reproducible spatiotemporal pattern. Neuroblast maps have been prepared from different stages of the early embryo (stages 9, 10 and 11, when the entire population of neuroblasts has formed), in which about 40 molecular markers representing the expression patterns of 34 different genes are linked to individual neuroblasts. In particular, a detailed description is presented of the spatiotemporal patterns of expression in the procephalic neuroectoderm and in the neuroblast layer of the gap genes empty spiracles, hunchback, huckebein, sloppy paired 1 and tailless; the homeotic gene labial; the early eye genes dachshund, eyeless and twin of eyeless; and several other marker genes (including castor, pdm1, fasciclin 2, klumpfuss, ladybird, runt and unplugged). Based on the combination of genes expressed, each brain neuroblast acquires a unique identity, and it is possible to follow the fate of individual neuroblasts through early neurogenesis. Furthermore, despite the highly derived patterns of expression in the procephalic segments, the co-expression of specific molecular markers discloses the existence of serially homologous neuroblasts in neuromeres of the ventral nerve cord and the brain. Taking into consideration that all brain neuroblasts are now assigned to particular neuromeres and individually identified by their unique gene expression, and that the genes found to be expressed are likely candidates for controlling the development of the respective neuroblasts, these data provide a basic framework for studying the mechanisms leading to pattern and cell diversity in the Drosophila brain, and for addressing those mechanisms that make the brain different from the truncal CNS (Urbach, 2003b).

The cephalic gap genes are expressed in large domains of the procephalon and play a crucial role not only in the patterning of the peripheral ectoderm, but also in regionalizing the brain primordium. The segmental organization of the Drosophila brain is based on the expression pattern of segment polarity and DV patterning genes. To see whether the cephalic gap genes respect the neuromeric boundaries segment polarity and DV patterning genes, and to provide a basis for studying their potential role in the formation or specification of brain precursor cells, the expression was studied of orthodenticle, empty spiracles, sloppy paired 1, tailless, huckebein, and hunchback in the developing head ectoderm, as well as in the entire population of identified NBs during stages 9-11 (Urbach, 2003b).

In the cellular blastoderm orthodenticle (otd) is expressed in an anterior, circumferential stripe and subsequently fades in the ventral region to become restricted to the procephalic ectoderm after gastrulation. In Otd/Engrailed (En) double labelling between stage 9 and 11, Otd expression in the pregnathal head is found to be confined to a large domain covering most of the antennal (the third neuromere) and preantennal (the second neuromere, termed 'ocular') neuroectoderm. Furthermore, Otd is detectable in all NBs delaminating from this domain (about 50 ocular and six antennal. NBs in the dorsal and most anterior region of the protocerebrum are Otd-negative, including most NBs of the labral neuromere (the most anterior neuromere). Thus Otd covers the NBs of the anterodorsal part of the antennal segment and most of the acron (which is equivalent to the ocular segment). Otd expression is also observed in cells along the dorsal midline of the head, as well as faint expression in neuroectodermal cells in the ventral part of the intercalary segment (the fourth neuromere - posterior to the antennal neuromere), from which the weakly Otd-positive Tv1 emerges (Urbach, 2003b).

tailless (tll) has been shown to be expressed in an anterior horseshoe-shaped stripe in the cellular blastoderm, which after gastrulation shows a region of high ('HL domain') and a region of low level of tll expression ('LL domain'), and at stage 9 covers most of the protocerebral neuroectoderm. Using a tll-lacZ line at stage 9 tll expression has been found in the developing brain in most protocerebral NBs (except the dorsoposterior ones). During stages 9-11 tll-lacZ expression expands in the protocerebral neuroectoderm beyond the En-positive head spot (hs). By stage 11 it is detectable in all protocerebral NBs. In addition, tll-lacZ is found in some ventral and dorsal deutocerebral NBs, indicating that tll is not exclusively confined to protocerebral progenitors (Urbach, 2003b).

During early neurogenesis in the trunk, empty spiracles (ems) is metamerically expressed in lateral ectodermal patches. In the head, it acts as a gap gene, which is expressed in a circumferential procephalon domain in the early cellular blastoderm. During gastrulation this circumferential stripe dissolves into three smaller ectodermal domains between the anterior part of the mandibular segment and the posterior part of the ocular segment; these domains are not in segmental register. During further development, the third domain splits into a mandibular/intercalary and an antennal component. All these domains contribute NBs to the brain. In addition to ems expression in the intercalary and antennal segments, and the corresponding trito- and deutocerebral neuromers, ems expression was also detected in a small neuroectodermal region. Finally, a further ems patch is located in the dorsoanterior procephalic ectoderm ('dorsal patch'), which becomes part of the labral ectoderm and does not appear to give rise to brain NBs. Thus, from stage 9 onwards, part of the antennal/ocular ems domain overlaps with the En-positive hs, and from stage 10/11 onwards these genes are also found to be co-expressed in the en hs-derived protocerebral NBs Ppd5 and Ppd8 (although en and ems expression also partly overlaps in the trunk ectoderm, a co-expression of both genes in trunk NBs is never seen). In contrast to earlier observations, showing that most of the tritocerebral NBs are included in the ems-expressing domain, only the dorsal Td6 was identified as Ems-positive. Ems protein is detectable in clusters of brain cells until the end of embryogenesis (Urbach, 2003b).

The sloppy paired (slp) locus contains the two related genes slp1 and slp2. slp1, which acts as a head gap gene, plays a predominant role in head formation, while slp2 is largely dispensable. In the trunk neuroectoderm, where slp1 has a function as a pair-rule and segment polarity gene, it is segmentally expressed in neuroectodermal stripes as well as in NBs of row 4 and 5. This segmental appearance of slp1 expression is found to be conserved in parts of the procephalon. In the blastoderm, Slp1 protein is detected in a large domain of the procephalon anlage, which subsequently diminishes in its anterior/ventral part. As a result, only the posterior half of the original slp1 domain remains as a circumferential ring ('head stripe') and gets separated from the anterodorsal part ('head cap'). To follow the dynamics in the Slp1 expression pattern, Slp1/En double labelling was examined during stages 8-11. The 'head stripe' corresponds to the slp1 stripe of the prospective mandibular segment, and the posterior part of the 'head cap' to the Slp1-positive stripe in the prospective antennal segment (slp1 as). At the beginning of gastrulation, a new Slp1 ectodermal spot in the anterodorsal procephalon is observed; this spot later becomes part of the labral ectoderm. In addition, at stage 9, three new ectodermal domains become detectable: one stripe anterior to the en intercalary stripe belonging to the intercalary segment, and two small spots in the region of the ocular segment (anterior to the en head spot). Except for the labral domain, the slp1 domains contribute NBs to the brain. Thus, slp1 is segmentally expressed in the procephalic neuroectoderm and subsets of brain NBs, resembling the situation in the trunk. At stage 11 patchy expression of Slp1 becomes detectable within the ocular and labral ectoderm and in some underlying ocular and labral NBs. Some of these NBs initiate slp1 expression after delamination; e.g. Pcv6 and Pcd2 delaminate at stage 9 and do not express slp1 before stage 11. Slp1 expression is observed in the brain until the end of embryogenesis (Urbach, 2003b).

huckebein (hkb), a terminal gap gene, is first expressed at the anterior and posterior blastodermal poles, where it is required for the specification of the endodermal anlagen, and later for the invagination of the stomodeum. After gastrulation, hkb becomes transiently expressed in a repetitive pattern in the trunk neuroectoderm and in eight, mainly intermediate, NBs per hemineuromere. In the procephalic region at the cellular blastoderm stage, hkb (Urbach, 2003b).

Expression is detected
in a centrally located stripe and a dorsal ectodermal spot. hkb in situ hybridization combined with anti-Inv antibody staining reveals that during stage 9/10 the hkb stripe covers most of the antennal ectoderm and reaches into the anterior region of the intercalary segment, and the hkb spot covers part of the ocular ectoderm. During stage 9 hkb transcript in the ocular spot becomes progressively restricted to the delaminating protocerebral NBs, Pcv7 and Pcd2, and remains strongly expressed in both NBs until stage 11. In the antennal domain during stage 10/11 the transcript becomes confined to three to five deutocerebral NBs. However, using a hkb-lacZ line (5953) the marker is expressed in all deutocerebral NBs at stage 10. At stage 11, hkb-lacZ was not detectable in Dd8 and Dd11, indicating that hkb is not a general deutocerebral NB marker. In the tritocerebrum, hkb is expressed only in Td6 (stage 10) and in Tv1, Td8. Thus, although expressed in a few trito- and protocerebral NBs, hkb expression appears to be mainly confined to the antennal neuroectoderm and NBs. Compared with the transcript, which becomes restricted to the NBs during stage 9-11, hkb-lacZ expression has a longer perdurance in the peripheral ectoderm and corresponding NBs. By stage 14, most of the hkb transcript has disappeared and is confined to some deutocerebral cells; hkb-lacZ is strongly expressed until the end of embryogenesis in deutocerebral, and at a lower level, in protocerebral cells, the putative progeny of the identified Hkb-positive brain NBs (Urbach, 2003b).

hunchback (hb) expression in the anterior half of the embryo falls below the limit of detection at the beginning of germ band extension, but accumulates during the extended germ band stage in the CNS, where it is transiently expressed in early, fully delaminated, trunk NBs (S1 and S2) and their progeny. Antibody staining reveals that, from stage 8 onwards, Hb protein is not detected in the head neuroectoderm, but is very dynamically expressed in brain NBs. At stage 9, only about half of the identified deuto- and protocerebral NBs show Hb protein at a detectable level, suggesting that Hb is not a general marker for early NBs. Correspondingly, it is found that Hb protein is also lacking in particular S1 and S2 NBs of the trunk. In some of the early brain NBs, Hb first becomes detectable at stage 10, after their delamination. For example, the early NBs Pcv9 and Pcd6 delaminate at late stage 8 but do not start Hb expression before stage 10. By stage 10, Hb is expressed in about 26 brain NBs, most of which delaminate between stage 9 and 10. In most of these NBs, Hb expression is progressively lost, but is observed in an increasing amount of progeny cells. At stage 11, it is confined to a small subpopulation of about five tritocerebral and four to six protocerebral NBs. Thus, as opposed to the trunk, hb expression in the brain is not limited to early NBs. Hb is expressed in the brain until stage 15, when it is detected in a few cells of the protocerebrum (Urbach, 2003b).

Taken together, among the cephalic gap genes, slp1 appears to respect segmental boundaries during early neurogenesis of the brain. By contrast, in the considered period of development (stage 9-11), the expression of ems, otd and tll does not seem to respect these borders, contradicting claims in previous reports. All three genes are expressed in NBs deriving from ectodermal domains that are part of two or three neighboring segments. For example, ems is expressed in a small number of NBs comprising about six posterior ocular and four anterior deutocerebral NBs (all of which derive from the same ems domain, except Dv3 and Pcv5), and one tritocerebral NB. Accordingly, ems mutants show defects in the intercalary, antennal, and the ocular segment (e.g., the en hs is missing). Considering that ems is expressed in only a few trito- and deuto-cerebral NBs it is remarkable that ems mutants show a deletion of the trito- and deuto-cerebrum. An explanation for this could be that ems expression, which during earlier development covers the neuroectoderm of the respective segments, possibly confers specific identities to the arising trito- and deuto-cerebral NBs. The lack of these NBs might be responsible for the loss of NB-specific gene expression, and (secondarily) for the gross morphological defects seen in the ems mutant brain. A similar proposal has been made to explain the brain defects that occur in buttonhead (btd) mutants, although btd is not expressed in NBs of the corresponding brain regions (Urbach, 2003b).

The expression was analyzed of the homeotic genes proboscipedia (pb) and labial (lab), both members of the Antennapedia complex and known to be expressed in the head ectoderm and in the brain after mid-embryogenesis. Antibody staining against Pb reveals that at stage 11, the protein is restricted to internal cells of the mandibular segment (presumably mesodermal cells) and to dorsal ectoderm of the maxillary and labial appendages. No Pb protein was detected in brain NBs (Urbach, 2003b).

lab has been described as being expressed in the posterior tritocerebrum at stage 14. Using an antibody, the expression of Lab protein during early neurogenesis was investigated. From stage 9 onwards, Lab is detected in the ectoderm of the intercalary segment, and presumably in a small part of the posteroventral antennal segment. At that stage, the only NB expressing Lab protein is Dv2. Double labelling against En reveals that at stage 11 Lab is expressed throughout the ectoderm of the intercalary segment. The Lab domain overlaps posteriorly with the en intercalary stripe (en is), indicating that posterior borders of lab expression and of the intercalary segment coincide. The character of the anterior border of the lab domain is less clear. Dorsally, it runs along the posterior border of the en antennal stripe (en as); ventrally, however, it reaches the anterior border of the en as. This suggests, that the anterior border of the lab domain is segmental in the dorsal region and parasegmental in the ventral region. Interestingly, also for scr and dfd, which are other members of the ANT-C, it has been reported that they initiate expression in a jagged stripe resolving into a pattern that is dorsally segmental and ventrally parasegmental. All NBs arising from the Lab-positive neuroectoderm express lab, among them all tritocerebral NBs and two ventral NBs, which are attributed to the deutocerebrum (Dv2 and Dv4) because they are located on the same anteroposterior level as the en-expressing Dv8 and Dd5 (Urbach, 2003b).

dachshund (dac) is involved in the development of the eye and the mushroom bodies where it is expressed already in the progenitor cells. Using an antibody, Dac expression was found in the trunk CNS not before stage 12; it is expressed in only two or three cells (not NBs) per neuromer. In the procephalon, Dac is already detected by stage 9 in a small area of the dorsal ocular neuroectoderm from which four Dac-positive NBs (Pcd4, Pcd8, Pcd9, Pcv9) delaminate. It has been suggested that the NBs delaminating from this Dac domain represent the progenitors of the mushroom body and co-express eyeless (ey). In disagreement with this, it was found that, at that stage, the co-expression of both genes is confined only to a small region of the Dac-positive neuroectoderm and to only one of the four identified Dac-positive NBs. As evidenced by Dac/Ey antibody double labelling, this NB (Pcv9) is one of the five Ey-positive brain NBs identified at stage 9. Until stage 11, the Dac-expressing ocular domain expands into the antennal segment and into the optic lobe anlage (now encompassing also the ectodermal region called 'para-MB neuroectoderm'), and a further spot appears in the clypeolabral ectoderm. At this stage, Dac protein can be observed in 13 protocerebral NBs and in the tritocerebral Tv2, but in no deutocerebral NBs. From stage 12 onwards, Dac becomes expressed in an increasing number of scattered cell clusters in the brain and ventral nerve cord (Urbach, 2003b).

eyeless (ey), which encodes a member of the Pax6 family of transcription factors, is a crucial regulator for eye development. It is expressed in the embryonic ventral nerve cord and brain and has been shown to be involved in the development of the mushroom bodies. Although it has been suggested that ey is expressed in the progenitor cells of the mushroom bodies, ey expression in the evolving NB pattern of the ventral nerve cord and brain has not been described to date. Using an Ey antibody (which principally shows the same pattern as ey mRNA in situ hybridization), Ey in the trunk is found to be expressed in segmentally reiterated ectodermal stripes and, at stage 11, in six NBs per hemineuromere. In the posterior pregnathal head segments (intercalary and antennal), a segmental expression of Ey appears to be fundamentally conserved. Ey/En double labelling reveals that the Ey-positive spots (and emerging NBs) in both segments are localized just anterior to their En-positive counterparts. Ey protein is detected, by stage 9, in five NBs: Pcv6, Pcv7, Pcv9, Pcd2 (which derive from an ocular ectodermal Ey domain) and Dv6 (which develops from a small ectodermal Ey spot in the antennal segment). By stage 10, ey becomes expressed in an intercalary ectodermal spot, from which, by that stage, the Ey-positive Td2 and (slightly later) Td1 delaminate, and in a second ocular ectodermal spot from which Ppd12 develops. In contrast to the intercalary and antennal ectodermal spot, Ey protein becomes depleted during stage 11 in the ocular domain. At late stage 11, Ey is found in 15 brain NBs, including two intercalary, three deutocerebral, nine ocular and one labral NB (Pad7). During subsequent development of the brain, the Ey expression pattern becomes complex, especially in the preantennal segments, but it appears to be mainly confined to the progeny of the identified NBs (Urbach, 2003b).

The second Drosophila Pax6 gene, twin of eyeless
(toy), has been shown to be expressed in the blastoderm in an
anterodorsal patch that represents the posterior region of the procephalon anlage, and later in the embryonic brain (including the mushroom body and visual system). At stage 9, Toy expression encompasses the dorsal ocular and the anterodorsal part of the antennal ectoderm. All NBs that delaminate from this part of the neuroectoderm express Toy. By stage 11, these include about 40 protocerebral NBs, all of which derive from the ocular ectoderm, except the putative labral Pav1, and two deutocerebral NBs. This pattern of expression in NBs closely matches the pattern that has been observed for otd. In some of these NBs expression of Toy protein is transient and ceases during stage 11 (e.g., Dd3). To determine whether both Pax6 genes, ey and toy, are co-expressed in identified NBs, toy in situ hybridization was performed combined with an Ey antibody staining. Interestingly, toy appears to be expressed only in those Ey-positive NBs deriving from the ocular segment; a co-expression was not seen in Ey-positive NBs of the trito- and deuto-cerebrum, or in Pad7. During later embryogenesis, Toy protein is expressed in cells of the proto- and deutocerebrum, as well as in the tritocerebrum, although no Toy-positive tritocerebral NBs were identified (Urbach, 2003b).

In order to establish further molecular markers that are specifically expressed in subsets of brain NBs, the expression of castor, Fasciclin 2, klumpfuss, ladybird early, POU-domain 1 gene, runt and unplugged were investigated for details of the spatiotemporal expression pattern of these genes in the neuroectoderm and brain NBs (stages 9, 10 and 11) (Urbach, 2003b).

In the trunk, the pair-rule gene runt is expressed in segmental domains of the ventral neuroectoderm and in five NBs of row 2 and 3 and two NBs of row 5. Runt has also been shown to be expressed in an anterodorsal region of the blastoderm, corresponding to the presumptive head region. En/Runt antibody co-labelling reveals that this Runt domain contributes to the ocular segment. In addition to the ocular segment, patches of runt expression are in the intercalary, antennal and clypeolabral ectoderm, and in subsets of protocerebral and deutocerebral NBs. At stage 11, the protein is expressed in a total of 23 brain NBs, some of which initiate Runt expression after delamination from Runt-negative ectoderm, and in a large number of postmitotic cells until the end of embryogenesis (Urbach, 2003b).

In the ventral nerve cord castor (cas), encoding a zinc-finger protein, has been shown to be expressed in 18 NBs per hemineuromere, including early (S1-S2) and late delaminating (S3-S5) NBs, and to be involved in cell fate control within NB lineages. In the procephalon, cas expression is not detectable before stage 10. It is dynamically expressed in the central and dorsal neuroectoderm of the ocular segment, in the median antennal segment, and, by stage 11, in the labral segment, which is surprising since cas is not expressed in the neuroectoderm of the trunk. A proportion of Cas-positive protocerebral and deutocerebral NBs are derive from these domains. Most NBs appear to delaminate from Cas-negative neuroectoderm, and start to express cas at the time of formation, or show a reproducible delay in the onset of cas expression. The latter may already have produced a part of their lineage, which likewise has been proposed for early trunk NBs (e.g. NB7-4). At late stage 11, Cas is expressed in about 60% of the total number of identified brain NBs (Urbach, 2003b).

Using an antibody against the cell membrane glycoprotein Fasciclin 2 (Fas2), it has been found that in the procephalic region Fas2 is first expressed by late stage 10 in an ectodermal patch at the border between the intercalary and antennal segment. Later it also covers the posterodorsal ocular neuroectoderm (including the optic lobe anlage) and part of the labral ectoderm. Fas2 is also detected in brain NBs emerging from the antennal and intercalary neuroectoderm, and at a low level in a few dorsal ocular NBs. It has been found that Fas2 controls proneural gene activity in the eye/antennal imaginal disc, raising the possibility that it functions likewise in the procephalic neuroectoderm. However, Fas2 expression in almost all identified brain NBs is initiated after delamination from Fas2-negative neuroectoderm, suggesting that Fas2 in the procephalic neuroectoderm is not involved in the regulation of proneural genes. It has been shown that Fas2 appears on the surface of neural somata prior to axon outgrowth; these neurons belong to 'fiber tract founder clusters' that pioneer the main axonal tracts in the brain. Considering position and time point of development, it is suggested that the identified Fas2-positive deuto- and trito-cerebral NBs (Tv1, Tv2, Td1, Td2, Td6, Td8; Dv2, Dd9, Dd11) are the precursors of the 'D/T fiber tract founder cluster' (Urbach, 2003b).

In the trunk, the zinc-finger transcription factor Klumpfuss (Klu) is expressed from stage 10 onwards in an increasing number of NBs, and at stage 11, almost all NBs (except NB2-3 and NB6-4) show nuclear Klu staining. The expression of Klu in the procephalon was analyzed using an antibody against Klu and the P-lacZ enhancer trap strain klu P212 which basically shows an identical expression pattern. Klu is not expressed in the neuroectoderm. Similar to the situation in the trunk CNS, Klu protein is first found at a detectable level at stage 9, in a subset of (about 17) brain NBs and at late stage 11 in almost all brain NBs. For most NBs, there is a significant delay between birth and onset of klu expression. Klu also appears to be expressed in ganglion mother cells, as was shown for the trunk (Urbach, 2003b).

ladybird (lb), a tandem of the homeobox genes ladybird early (lbe) and ladybird late (lbl), both of which encode transcription factors, show a similar expression pattern, with lbe activity slightly preceding that of lbl. At stage 11, both genes are expressed in segmental repetitive patches in the laterodorsal trunk ectoderm and specifically in one NB per hemineuromere, the lateral NB 5-6. Using an antibody against Lbe the protein is first observved by stage 10 in three small procephalic patches in the labral, ocular and antennal ectoderm, and at stage 11 in an additional patch of the intercalary ectoderm. Lbe is selectively expressed in only four brain NBs on either side: one in the tritocerebrum (Td4), one in the deutocerebrum (Dd7) and two in the protocerebrum (Ppv3, Pcv8). Wg/Lbe double labelling demonstrates that Lbe and Wg expression are colocalized in the intercalary, antennal and labral ectoderm, and in Td4 and Dd7; remarkably, the ocular Lbe-positive domain and corresponding NBs (Ppv3 and Pcv8) are Wg negative. Lbe protein is detected in the progeny of the identified brain NBs until the end of embryogenesis (Urbach, 2003b).

The two closely related Drosophila POU-domain genes, pdm1 (nubbin) and pdm2, are co-expressed in the developing CNS (before stage 13) and have been shown (at least with respect to the specification of the first ganglion mother cell of the truncal NB4-2) to be functionally redundant. pdm1 is expressed in the trunk neuroectoderm during the first and second wave of NB segregation (stage 8/9), and transiently in most NBs at stage 10 and 11. In the procephalon the expression of the Pdm1 protein is highly dynamic. Until stage 10, Pdm1 is roughly restricted to the neuroectoderm of the antennal and ocular segments. Later, it is also found in the intercalary and labral ectoderm. At stage 9, NBs derived from Pdm1-positive neuroectoderm appear to be Pdm1 negative and initiate pdm1 expression at stage 10 or stage 11. At late stage 11, approximately one half of the brain NBs (about 52 NBs) express pdm1, including most deuto- and trito-cerebral NBs, as well as central ocular NBs and part of the labral NBs (Urbach, 2003b).

Expression of the homeodomain gene unplugged (unpg) in the trunk starts at stage 8 in the ventral midline and becomes detectable in NBs of the ventral nerve cord at late stage 11. Using an unpg-lacZ line, unpg expression is observed in the head at stage 9 in a large domain encompassing the intercalary, antennal and most of the ocular ectoderm. Until stage 11, the expression is gradually lost in the intercalary ectoderm, but upregulated in the dorsal part of the antennal and adjacent ocular ectoderm. In contrast to trunk NBs, which have already divided several times before expressing unpg at late stage 11, unpg-lacZ is weakly expressed already at stage 9 in all deutocerebral and almost all protocerebral NBs. At late stage 11, it is strongly expressed in almost all deutocerebral NBs (except for some ventral ones), and in some ocular NBs close to the deutocerebral/ocular border. Until the end of embryogenesis, unpg expression is observed in the putative progeny cells of the unpg-lacZ-positive deuto- and protocerebral NBs (Urbach, 2003b).

For thoracic and abdominal segments, each NB acquires a unique identity, which corresponds to a particular position in the neuroectoderm and (upon delamination) in the subectodermal NB layer, to a certain time point of its delamination, and to the combination of genes expressed. Descriptions of gene expression has provided an important basis for the elucidation of mechanisms controlling cell fate specification during early neurogenesis in the trunk region. In contrast to the truncal CNS, in which the segmental organization is obvious and the composition of the neuromeres is almost identical, the brain neuromeres are much more diverse and complex. Accordingly, information on identified brain cells and their gene expression is hardly available so far, and thus essential tools for investigating the mechanisms underlying pattern formation and cell diversity in the brain are lacking (Urbach, 2003b).

Comparison of the combination of markers expressed in individual NBs as well as their relative position within the NB layer of each segment suggests that several NBs exist in the brain that are serially homologous to NBs in the ventral nerve cord (VNC). This mainly applies to the posterior brain (deuto- and trito-cerebrum), which is less derived than the anterior brain (protocerebrum). For example, according to these criteria, NB5-6 in all abdominal, thoracic and gnathal neuromeres would be serially homologous to Td4 in the tritocerebrum and to Dd7 in the deutocerebrum. These NBs exhibit a similar posterodorsal position within the respective neuromer immediately anterior to the En-positive NBs, and are the only NBs which specifically co-express the following molecular markers: lbe (which is generally expressed in only one NB per hemisegment), wg, gsb-d, slp1 (except Td4), msh, cas, seven-up (except Td4), pdm1, klu and asense. Furthermore, some of the daughter cells of Td4 and NB5-6 co-express ladybird and the glia-specific marker reversed polarity. The existence of serially homologous NBs is intriguing since the number of NBs in the tritocerebrum and deutocerebrum is considerably reduced, the timecourse of neurogenesis within the brain and VNC is different (especially in the tritocerebrum the development of NBs is significantly delayed), and the development of head segments (and consequently of brain neuromeres) has been assumed to be differently regulated (Urbach, 2003b).

In the VNC, serially homologous NBs that express the same combination of molecular markers give rise to almost identical cell lineages, suggesting that similar regulatory interactions take place during the development of these NBs and their cell lineages. However, some of the serially homologous VNC lineages have been shown to include a subset of progeny cells that specifically differ between thoracic and abdominal neuromeres. It is expected that such segment-specific differences are even more pronounced among serially homologous lineages within the brain and between the brain and VNC. Differences in the combination of marker genes expressed by putative serially homologous NBs may point to candidate genes conferring segment-specific characteristics to their lineages. Thus, unravelling the lineages of serially homologous NBs and the genetic network that controls their development will help to elucidate how region-specific structural and functional diversity in the CNS evolves from a basic developmental ground state (Urbach, 2003b).

Animals from flies to humans are able to distinguish subtle gradations in temperature and show strong temperature preferences. Animals move to environments of optimal temperature and some manipulate the temperature of their surroundings, as humans do using clothing and shelter. Despite the ubiquitous influence of environmental temperature on animal behaviour, the neural circuits and strategies through which animals select a preferred temperature remain largely unknown. This study identified small set of warmth-activated anterior cell (AC) neurons located in the Drosophila brain, the function of which is critical for preferred temperature selection. AC neuron activation occurs just above the fly's preferred temperature and depends on dTrpA1, an ion channel that functions as a molecular sensor of warmth. Flies that selectively express dTrpA1 in the AC neurons select normal temperatures, whereas flies in which dTrpA1 function is reduced or eliminated choose warmer temperatures. This internal warmth-sensing pathway promotes avoidance of slightly elevated temperatures and acts together with a distinct pathway for cold avoidance to set the fly's preferred temperature. Thus, flies select a preferred temperature by using a thermal sensing pathway tuned to trigger avoidance of temperatures that deviate even slightly from the preferred temperature. This provides a potentially general strategy for robustly selecting a narrow temperature range optimal for survival (Hamada, 2008).

Although the physiology of all cells is affected by temperature, the expression of temperature-activated members of the transient receptor potential (TRP) family (thermoTRPs) can make cell excitability highly temperature-responsive (Dhaka, 2006). ThermoTRPs are cation channels with highly temperature-dependent conductances that participate in thermosensation from insects to humans. The Drosophila TRP channel dTrpA1 promotes larval heat avoidance (Rosenzweig, 2005) and can be activated by warming in ooctyes (Viswanath, 2003). This study addressed whether dTrpA1 contributes to the selection of a preferred temperature in the adult fly. When allowed to distribute along a thermal gradient for 30 min, wild-type D. melanogaster adults prefer ~25°C, their optimal growth temperature. Compared to wild-type controls, dTrpA1 loss-of-function mutant animals showed increased accumulation in the warmest (28-32°C) regions of the gradient, but not in the coolest (18-22°C) regions. A dTrpA1 genomic minigene rescued the phenotype. Animals heterozygous for dTrpA1 loss-of-function mutations also preferred slightly elevated temperatures. Thus, dTrpA1 function is important for determining thermal preference and specifically contributes to avoidance of warm regions (Hamada, 2008).

If dTrpA1 was involved in thermotransduction, it should regulate the warmth responsiveness of thermosensors. As the identity of the adult Drosophila thermosensors was unknown, dTrpA1 protein expression was examined (using anti-dTrpA1 antisera). dTrpA1 expression was detected in three sets of previously uncharacterized cells in the brain: lateral cell (LC), ventral cell (VC) and AC neurons. dTrpA1 was also detected in the proboscis, but ablation studies detected no contribution of the proboscis to warmth avoidance. To focus on the neurons that contribute to thermal preference, where the rescuing dTrpA1 minigene restored dTrpA1 expression was examined; dTrpA1 expression was restored specifically within AC neurons, but not LC or VC neurons. This suggested that dTrpA1 expression in AC neurons (two pairs of neurons at the brain's anterior) sufficed to restore thermal preference and that AC neurons might act as thermosensors (Hamada, 2008).

Temperature responsiveness of AC neurons was examined using the fluorescent calcium indicator G-CaMP. When exposed to increasing temperature, AC neurons showed robust increases in G-CaMP fluorescence, reflecting warmth-responsive increases in intracellular calcium. Ten out of the 27 AC neurons imaged had fluorescence increases between 4% and 39%, with a mean increase over baseline among these cells of 15%. The average temperature at which fluorescence increases were initially observed was 24.9°C, compatible with AC activation as temperatures rise above preferred. In contrast, none of the 21 dTrpA1 mutant AC neurons imaged had fluorescence increases. As a control that mutant AC neurons remained physiologically active, it was confirmed that they showed robust responses on potassium chloride addition. Notably, AC responses did not depend on an intact periphery, since all G-CaMP studies were performed using isolated brains from which peripheral tissues had been removed. These observations identify AC neurons as warmth-activated, dTrpA1-dependent thermosensors (Hamada, 2008).

AC neurons project towards several brain regions, including the antennal lobe. The antennal lobe is implicated in cockroach thermosensation, but has been studied exclusively for olfaction in Drosophila. So far, 11 of the ~50 antennal lobe glomeruli remain unassociated with identified olfactory receptors. AC neurites elaborated within two such unassociated glomeruli, VL2a and VL2p. Thus the Drosophila antennal lobe contains both thermosensory and olfactory neuron processes. VL2a is also innervated by Fruitless-expressing neurons implicated in pheromone transduction, suggesting that even individual glomeruli receive multi-modal sensory information. AC processes also branched within the subesophageal ganglion and superior lateral protocerebrum, although these target regions are less defined than in the antennal lobe. These regions have been previously implicated in processing other types of sensory input (Hamada, 2008).

As dTrpA1 expression in AC neurons seemed sufficient to restore normal thermal preference, whether such expression was also necessary was also examined. dTrpA1 was knocked down selectively in AC neurons using tissue-specific RNA interference targeting dTrpA1 controlled by dTrpA1SH-GAL4, a promoter expressed in AC but not LC or VC neurons. Consistent with the importance of dTrpA1 expression in AC neurons in thermal preference, AC knockdown increased the fraction of animals present in the 28-32°C region compared to controls. Similar results were obtained when dTrpA1 expression was knocked down using a broad neuronal promoter (Appl-GAL4). All knockdowns were assessed by dTrpA1 immunohistochemistry. dTrpA1 knockdown with the general cholinergic neuron promoter Cha(7.4)-GAL4 eliminated detectable dTrpA1 expression in AC (and LC and VC ) neurons, decreasing warmth avoidance. In contrast, dTrpA1 RNAi expressed using Cha(1.2)-GAL4 -- which is expressed in many brain cholinergic neurons but not AC neurons -- did not disrupt warmth avoidance. Taken together, these data suggest that dTrpA1 expression in AC (but not LC or VC) neurons is both necessary and sufficient for normal thermal preference behaviour. Whether LC and VC neurons participate in other warmth-activated responses is unknown (Hamada, 2008).

The identification of an internal sensor controlling temperature preference conflicts with the established view that Drosophila sense moderate warming using thermosensors in the third antennal segment. The effects were tested of surgically removing either one third antennal segment and arista (unilateral ablation) or both (bilateral ablation). Both unilateral and bilateral ablation increased the fraction of animals in cool (18-22°C), but not warm (28-32°C), regions. Thus these tissues were dispensable for warmth avoidance, but essential for cool avoidance. When dTrpA1 mutants were subjected to bilateral ablation, they accumulated in both cool and warm regions: the fraction between 18-22°C did not differ from wild-type ablation animals; the fraction between 28-32°C did not differ from non-ablated dTrpA1 mutants. Thus dTrpA1-expressing cells and antennal cells function additively to set preferred temperature, promoting avoidance of elevated and reduced temperatures, respectively (Hamada, 2008).

These data are consistent with warmth activation of dTrpA1 serving as the molecular basis of warmth sensing by AC neurons. As thermal activation of mammalian TRPA1 proteins is controversial, whether dTrpA1 could act as a molecular sensor of warming in the fly was tested. Indeed, misexpression of dTrpA1 throughout the fly nervous system (using C155-GAL4) caused a dramatic phenotype not observed in controls: heating these flies to 35°C for 60 s caused incapacitation, an effect reversed on return to 23°C. Similar effects were observed using electrophysiology, with moderate warming (above ~25°C) triggering a barrage of excitatory junction potentials at the neuromuscular junction. These data strongly support dTrpA1 acting as a molecular sensor of warming. The ability of dTrpA1 mis-expression to confer warmth activation also suggests that dTrpA1 can be used as a genetically encoded tool for cell-specific, inducible neuronal activation. dTrpA1 might be particularly useful in tissues such as the fly brain where thermal stimulation is easier to deliver than the chemical or optical stimulation that controls other tools for modulating neuronal activity (Hamada, 2008).

To test whether warmth activation is a property of other insect TrpA1s, the malaria mosquito Anopheles gambiae TrpA1 (agTrpA1) was examined. dTrpA1 is warmth-activated when expressed in Xenopus laevis oocytes. agTrpA1 also showed robust warmth activation. These currents were specific, they were not observed in uninjected oocytes and were inhibited by ruthenium red (which antagonizes other TRPs). Similar to mammalian thermoTRPs, both dTrpA1 and agTrpA1 showed outward rectification. Closely related TrpA1s are present in the flour beetle Tribolium castaneum and in disease vectors such as Pediculus humanus corporis (body lice), Culex pipiens (common house mosquito) and Aedes aegypti (yellow and dengue fever mosquito) which use warmth-sensing for host location and habitat selection. Such insect TrpA1s constitute potential targets for disrupting thermal preference and other thermosensory behaviours in agricultural pests and disease vectors (Hamada, 2008).

Environmental temperature affects the physiology of all animals. Increasing temperatures associated with climate change are linked to poleward redistributions of hundreds of species including insects, fish, birds and mammals, AC neurons are internal. As a ~1 mg fly is readily penetrated by ambient temperature variations, such an internal sensor should monitor environmental temperature effectively. dTrpA1 activation seems to be critical for AC neuron activation, suggesting that dTrpA1 threshold and expression changes could modulate thermal preferences. More speculatively, changes in insect TrpA1 function and expression could facilitate movements into novel environments or development of novel behaviours such as host seeking (Hamada, 2008).

Although effects of environmental temperature on behaviour are ubiquitous, the mechanisms animals use to seek out optimal temperatures are largely unknown. AC neurons become active as temperatures rise above the preferred temperature, suggesting that they may function as 'discomfort' receptors that, together with putative antennal cool receptors (similar to those described in other insect antennae), repel the fly from all but the most optimal temperatures. Notably, mice lacking the cool-activated channel TRPM8 prefer abnormally cool temperatures, whereas mice lacking heat-activated TRPV4 prefer warmer temperatures, indicating that similar strategies may be used in mammals (Hamada, 2008).

All organisms need to sense and adapt to changes in nutrient
levels and nutrient demand. In vertebrates, this is achieved
through close monitoring of available nutrients by sentinel tissues
such as the gut, adipose tissue, and the pancreas, which,
in turn, signals the nutritional status to the brain, ultimately leading
to changes in metabolism and food intake. In the brain, nutrient-sensing neurons also respond directly to fuel-related stimuli like glucose, fatty acids, or amino
acids, engaging neurophysiological responses that control
energy intake. However, the neurochemical
identity of these neurons and the molecular sensors
used are in many instances unknown (Bjordal, 2014).

The complexity of the vertebrate brain presents a challenge to
understand the integration of nutrient signals and the molecular
and cellular mechanisms of neuronal nutrient sensing. A possible
alternative is to use genetically tractable organisms with simpler
brain structures like the fruit fly Drosophila.
Indeed, Drosophila recapitulates many of the hallmarks of
peripheral and central nutrient sensing seen in mammals. At
the periphery, a main nutrient sensor located in fat cells signals
to the brain and controls the release of Drosophila insulin-like
peptides (Dilps). In conditions of nutrient restriction,
the drop in general insulin signaling affects growth of peripheral
tissues as well as the function of specific neuropeptides such as
the Drosophila orthologs of neuropeptide Y, called NPF and sNPF, leading to changes in feeding behavior (Bjordal, 2014).

Recent reports also point to the presence of central nutrient
sensors regulating food intake. Experiments made on tasteless
animals have revealed that mice and flies are able to evaluate
the caloric content of carbohydrates independently of sweet
tasting. Interestingly, the Drosophila fructose receptor Gr43a is expressed
in specific neurons of the adult brain and controls
feeding according to circulating hemolymph fructose. Therefore, central fructose-sensing neurons could represent a new type of sensor for carbohydrates, although the
cellular and molecular mechanisms by which GR43a acts to
regulate food intake remain elusive (Bjordal, 2014).

Besides sugar, adult flies sense changes in dietary amino acid
levels, and a deprivation in amino acid induces a change in their
feeding preference toward amino acids. The downstream effector of the target of rapamycin (TOR) pathway, S6-kinase, and the neurotransmitter serotonin are involved in this regulation.
However, the detailed molecular mechanisms and the cellular
identity of such amino acid sensor are unknown (Bjordal, 2014).

One aspect of amino acid sensing concerns the necessity to
provide essential amino acids (EAAs) that cannot be synthesized
or stored. Earlier experiments in rodents have demonstrated that
animals rapidly evaluate the lack of one essential amino acid in
the food and initiate a series of drastic changes in behavioral
strategies, starting with food avoidance. Injection of imbalanced
amino acid mixes in defined areas of the rodent brain is sufficient
to trigger a reduction in food intake, suggesting that the sensor
for EAA deficiency (EAAD) is located in the brain. Additionally, mice with a mutation in the gene encoding the conserved GC nonderepressing 2 (GCN2) kinase do not
reject the imbalanced diet, indicating a role for this cell-based amino acid sensor in
triggering the EAAD response. The neural circuitry involved in this behavior remains
uncharacterized (Bjordal, 2014).

This study has identified a neural circuitry involved in amino acid
sensing and the control of feeding behavior in the Drosophila
larval brain. Drosophila larvae were shown to reduce food intake
when encountering an EAAD, and it was demonstrated that amino acid
sensing takes place in a limited number of dopaminergic (DA)
neurons. Calcium imaging in live brain show that DA cells are
rapidly and reversibly activated by EAAD in a GCN2-dependent
manner. Finally, using tissue-targeted genetic loss and gain-of-function
tools, it was demonstrated that EAAD-induced food avoidance
involves a GCN2-dependent inhibition of GABA signaling
in dopaminergic neurons. This demonstrates the existence of a
dopaminergic circuitry providing homeostatic control on feeding
through a central amino acid sensing mechanism (Bjordal, 2014).

Drosophila larvae reduce their food intake on EAAD diet. This behavior does not rely on smell or taste because it can be specifically mimicked or suppressed by interfering with amino acid sensing in the brain. In addition, ex vivo brain imaging demonstrates that DA
cells directly and rapidly activate in response to EAAD. The fast kinetics of
the response observed in DA cells suggests that uncharged tRNA levels are
instantly linked to variations in intracellular amino acid concentrations and translated into changes in GCN2 activity. GCN2 activation leads to several cellular
responses, including a block in translation initiation through eIF2a phosphorylation
and the consequent activation of a specific transcription program in which the ATF4 transcription
factor plays a key role. This study demonstrates that dATF4 in DA cells is required for the rejection of EAAD food. Given the very fast kinetics of neuronal
activation, it is unlikely that transcription participates in acute
EAAD avoidance. Using genetic interactions and ex vivo
calcium imaging, it was shown that EAAD-induced feeding inhibition
requires the repression of GABA signaling by dGCN2
activation in DA cells. In addition, bioluminescence resonance energy transfer (BRET) analysis demonstrates that ATF4 and GABA(B)R1 directly interact in living cells. These data are supported by observations made in
rodents indicating that suppression of GABAergic inhibition
contributes to EAAD-induced food avoidance. A model is proposed whereby, in response to EAAD, activation of dGCN2 induces dATF4-mediated
GABA signaling inhibition, dopamine release, and food rejection (Bjordal, 2014).

TOR signaling couples amino acid availability with the systemic
control of growth in fat body cells and ecdysone production
in the larval ring gland. Interestingly, TOR inhibition in DA cells does not attenuate EAAD-induced food avoidance. Similarly, rapamycin
injection in the antero-piriform complex of rodent brain does
not alter EAAD-induced feeding inhibition, supporting
the notion that GCN2, but not TOR signaling, is the
sensor for EAAD response. How independent these pathways
are is still an open question. Work in yeast suggests that TOR
acts upstream of GCN2. Such functional epistasis has not been established in
metazoan cells, and the present data suggest that the two pathways
operate independently in vivo (Bjordal, 2014).

Not all DA neurons are activated by EAAD. Using live imaging,
this study repeatedly observed that the DM1 and DL1 cluster, but not
the DL2 cluster, are activated by EAAD. Interestingly, this cluster
was recently implicated in olfactory reward-driven feeding, indicating subfunctionality among different dopamine circuits. Using subtraction analysis, it was possible to show that only three neurons in the DL1 cluster are responsible for EAAD-induced food avoidance. Nevertheless, additional DA
cells are activated by EAAD, suggesting that they could contribute
to other EAAD-induced behaviors. Indeed, EAAD induces
long-term effects such as the development of a learned aversion to a deficient
or imbalanced food and memory for the place associated with EAAD food. Hence, activation
of other DA cells by EAAD may contribute to these additional behaviors (Bjordal, 2014).

This work demonstrates a direct role of DA in nutrient sensing and food rejection
in flies; however, its role in aversive learning is well established. The activity
of the PPL1 cluster of DA neurons in the adult fly brain can produce aversive
memory when paired with an odor. Distinct DA neurons in the PPL1 cluster provide
motivational control over memory expression, suggesting that
these neurons constitute a dopaminergic circuitry that regulates
the internal motivational state of hunger and satiety. Direct lineage tracing remains to be done, but the
D0 and C1 Gal4 drivers targeting the subdomains of the larval
DL1 cluster also target the PPL1 cluster in the adult brain, suggesting that DL1 and PPL1 cells may be related.
Therefore, DA signaling in the DL1/PPL1 cluster could act as a
general satiation signal, reducing food intake and abolishing
appetitive performance (Bjordal, 2014).

The dopaminergic circuitry is known for its role in the motivational
control of feeding. This study has shown that it also plays a key
role in the homeostatic regulation of food intake. In light of recent
studies showing that metabolic hormones also exert their effect
on the dopamine reward circuit, the emerging picture is that dopamine
is a central player in the regulation of food intake through
the integration of nutrient sensing and motivational drives (Bjordal, 2014).

Animals exhibit innate behaviours to a variety of sensory stimuli including olfactory cues. In Drosophila, one higher olfactory centre, the lateral horn (LH), is implicated in innate behaviour. However, structural and functional understanding of the LH is scant, in large part due to a lack of sparse neurogenetic tools for this region. This study generated a collection of split-GAL4 driver lines providing genetic access to 82 LH cell types. These were used to create an anatomical and neurotransmitter map of the LH and link this to EM connectomics data. It was found that ~30% of LH projections converge with outputs from the mushroom body, site of olfactory learning and memory. Using optogenetic activation, this study identify LH cell types that drive changes in valence behavior or specific locomotor programs. In summary, this study has generated a resource for manipulating and mapping LH neurons, providing new insights into the circuit basis of innate and learned olfactory behavior (Dolan, 2019).

Previous work has classified LH cell types with either electrophysiology or calcium imaging. However, due to the lack of sparse driver lines, specific genetic access was not possible for most LH cell types. Two recent studies have identified more driver lines labelling LH neurons but these are broad and suitable mostly for electrophysiology and dye-filling identification of individual cells. This study has generated split-GAL4 lines that enable anatomical analyses and specific neurogenetic control of LH cell types. These reagentswere used to generate an atlas of identified LH neurons, defined their major neurotransmitter and polarity; classifying most cell types into LHONs, LHLNs and LHINs (Output, Local and Input)). Connectomic analysis of neural circuits in EM volumes is providing an unprecedented window into the structure of neural circuits, especially in Drosophila. To facilitate connecting these genetic tools with EM data this study has provided traced backbones of neurons from many identified LH cell types in a whole-brain EM volume, in addition to light-level single-neuron labelling. These resources will allow future studies to correlate functional and connectomic data to determine how the LH generates olfactory behaviour (Dolan, 2019).

This study used driver lines to identify major output zones and convergence sites of LH neurons. The diversity of inter- and intra-regional connections demonstrates the complexity of the LH and the innate olfactory circuitry, even in the relatively 'simple' brain of Drosophila. In terms of output, anatomical analysis identified the superior lateral protocerebrum (SLP) as the next major site of olfactory processing, although LHONs projected to many other brain regions, with a broader range than MBONs. Across identified cell types, no LHONs were identified that project to the ventral nerve cord, suggesting at least one more layer of processing before motor output. Within the LH, previous studies characterized one population of GABAergic local neurons. Other than these and MB associated neurons however, the neurotransmitter profiles of neurons in the protocerebrum, were unknown. This study identified populations of LH neurons that were cholinergic, GABAergic and/or glutamatergic. LHONs, LHINs and LHLNs were clustered into groups based on the sign of their neurotransmitter. Several distinct populations of both GABAergic and glutamatergic cells were found that were local to the LH, some potentially interacting with many LHON dendrites. This indicates that both lateral excitation and inhibition may exist in the LH. It was also found that the LH integrates visual, gustatory, thermosensory and mechanosensory input in a restricted, ventral region (Dolan, 2019).

The LH and MB are thought to mediate innate and learned behaviour respectively, yet their interactions remain little understood. Several MBONs project to the LH and this study has identified potential downstream targets. Indeed, one prediction (connectivity from MBON-α2sc to PD2a1/b1 neurons) has been validated and shown to be necessary for innate and learned behaviour (Dolan, 2018). In addition this study found that AD2b2 neurons extend their dendrite outside the LH and receive MB input. Previous studies have identified three instances of MB and LHON axonal convergence, however it was unclear if this was the case for all LHONs. This study systematically compared overlap for all the LHONs identified in the split-GAL4 screen. ~30% of LHONs were found to converge with MB-associated neurons, several of which appear placed to interact with more than one DAN and/or MBON. Therefore while this is a significant circuit motif, it does not explain all LHON projections. AD1b2 interact extensively with three MBONs in a unique axoaxonic integration motif, and stimulation of AD1b2 neurons drives approach behaviour when stimulated. Although the MB and LH both receive input from the AL, the coding logic of these two regions is strikingly different and the new tools described in this study will greatly facilitate studies of how these regions together orchestrate behaviour (Dolan, 2019).

To apply these reagents experimentally, cell type specific optogenetic activation were used, and several LH neurons were identified whose activation drives behaviour. Two cell types (one LHON and one LHLN) were identified that drive aversion, one LHON which drives attraction and several LHONs whose stimulation leads to changes in motor behaviours. The optogenetic screen identified only 3/50 LH cell types that could alone consistently drive changes in valence. This was a lower proportion than in the MB, where 6/20 MBON cell types tested were implicated solely in attraction and aversion rather than specific motor parameters (Dolan, 2019).

In total, the split-GAL4 lines covered 82 different cell types in the LH, although 24 of these were consistently colabeled in split-GAL4 lines. In addition, five anatomical cell types (PV4a1:5) occur in overlapping but disjoint split-GAL4 lines. Anatomical analysis of single neurons labeled with either stochastic genetic labelling or dye fills during recording identified ~165 different 'core' cell types in the LH (Frechter, 2019), that is cell types that seemed to have a majority of their dendrite within the LH and in the vicinity of olfactory uniglomerular PNs. Core LH cell types excluded LHINs and any neurons with a low fraction of their dendritic arbor overlapping the PN terminals bounded by the LH (Frechter, 2019). To calculate coverage of this study compared to these datasets, the same definition was used to categorize LH cell types identified in split-GAL4 lines as core or non-core. For LHON and LHLN cell types (68 in total), 63 were classified as core LH cell types. Based on this definition, the split-GAL4 collection covers 63/165 or ~38% of known core LH cell types (Dolan, 2019).

For the five cell types not defined as core, connectomic analysis indicates that they receive substantial input from multiglomerular PNs and/or some level of input from canonical uniglomerular PNs (Dolan, 2019).

Although this 38% is lower than estimates of coverage for the MB, which exceeds 80%, it is similar to the estimated coverage achieved to date (30-50%) for split-GAL4 lines targeting descending interneurons from the brain to VNC (Namiki, 2018). There are several possible hypotheses for these differences. The first is simply that there appear to be fewer MB cell types, for example 23 MBONs, than LH or descending interneurons. The second is that the dense, compartmental arborization of most MB neurons makes them easier to identify in lines containing many cell types, which are the starting point for split-GAL4 screens. Finally, it may well be that neurons in different brain regions have more or less distinct transcriptomic and epigenetic profiles and that this impacts the genetic isolation of their constituent cells. It is also emphasized that full and comprehensive reconstruction of all these neuronal classes from whole brain EM data will likely reveal additional cell types not discovered in these annotations of genetic data(Dolan, 2019).

This split-GAL4 driver line resource will now allow investigators to manipulate many LH neurons with cell type specificity. Driver lines can be used for imaging, functional activation or silencing experiments with genetically-encoded optogenetic or thermogenetic tools. However, the final expression pattern of any driver line also depends on the insertion site of the effector transgene; expression patterns should be verified for effectors at locations other than attP18 (the site used during this screening) (Dolan, 2019).

In the course of this work this study has produced large-scale anatomical maps of overlap between different populations of LH and MB neurons. This is a rapid approach to generate hypotheses for synaptic connectivity and circuit motifs. However, all potential synaptic interactions are subject to both registration error and biological variability across different brains. While several highly overlapping pairs with EM reconstruction were validated, even neurons that are very close in space may not be synaptically connected and this must be confirmed with connectomics or physiology (Dolan, 2019).

During behavioural studies, this study focused only on phenotypes that were consistent across multiple 'ideal' split-GAL4 lines for the same cell type. Indeed one of the four PD2a1/b1 lines tested had an aversive phenotype when activated, contrary to previous data (Dolan, 2018). This is most likely due to off-target expression in the VNC, as described (Dolan, 2018) and it is noted that stimulation of PD2a1/b1 neurons, using a different split-GAL4 line, in flying animals drove attraction. Despite this, not all differences between split-GAL4 lines ostensibly targeting the same cell type should be discarded. Some of the differences in phenotype between split-GAL4 lines targeting the same cell type could be due to differences in expression strength or simply experimental variation during a large-scale screen (that was not designed to identify small effect sizes). Even within an anatomically and genetically defined cell type, neurons can have different connectivities (Dolan, 2018). Therefore it is quite possible that this study has discounted viable phenotypes in this analysis and subsequent replication experiments. Finally, it also possible that the relatively low number of phenotypes in optogenetic activation screen is because activation of just one of >150 LH cell types is often too selective to produce strong behavioural consequences, especially in the absence of an olfactory stimulus. Future studies will be able to combine split-GAL4 lines to control multiple LH cell typesor use transsynaptic labelling to control large populations postsynaptic to specific cell types (Dolan, 2019).

This paper has directly demonstrated the existence of a diversity of genetically defined cell types with highly stereotyped dendritic arbours in the LH. Additionally, it was demonstrated that stimulation of LH neurons can drive valence or motor behaviours. While gain-of-function experiments do not alone demonstrate the role of the LH in innate behaviour, when combined with published data there is now clear anatomical, functional and behavioural evidence that the LH mediates instinctive olfactory responses. Firstly, anatomical and functional experiments demonstrate stereotyped connectivity between PNs and LH cell types between animals, implying a role in the generation of innate behaviour in contrast to the nearly random connectivity between PNs and the MB. Neuronal silencing experiments demonstrate that abolition of KC neurotransmission leads to a silencing of memory and a reset to innate olfactory responses . In addition to this work, two contemporaneous studies from the Jenilia group has used a subset of the new split-GAL4 lines to interrogate the role of this region in specific learned and innate olfactory behaviours. While Dolan (2018) shows that PD2a1/b1 LHONs are required for both innate attraction and aversive memory recall, the memory recall phenotype seems best understood as the modulation by a mushroom body output pathway of a hardwired pathway required for naive behaviour (Dolan, 2019).

Given the extensive interactions between LH and MB identified in this study it is proposed that these 'horizontal' pathways (e.g., MBONs projecting to LH, LHON to DAN) orchestrate additional functions such as memory retrieval, provide categorical information and MB modulation. It is suggested that LH-to-MB information flow (e.g, via LHON-to-DAN synapses) may also explain why some MBONs exhibit valence-specific responses, a hypothesis which can now be tested using the split-GAL4 lines developed in this study (Dolan, 2019).

The tools described in this study provide a critical resource for cell type specific dissection of the LH. Stimulation of AV1a1 and PV4a1:5 drive avoidance while silencing AV1a1 abolishes the response to geosmin (an ecologically relevant aversive odour) in an oviposition assay (Chen, 2018). This suggests AV1a1 LHONs may be a major pathway for ethologically-relevant aversion in the fly brain and that PV4a1:5 interneurons may pool other inputs to drive aversion via this pathway (Dolan, 2019).

This study also found that AD1b2 LHONs drive approach behaviour when activated. These cholinergic neurons project axons to the Superior Medial Protocerebrum (SMP) and have a distinctive additional dendritic projection extending out to the SMP/SIP. AD1b2 integrates MBON input in both an axodendritic and axoaxonic manner, including two MBONs which bidirectionally drive valence behaviour. Interestingly, previous work has identified another LHON (PD2a1/b1) involved in innate attraction. PD2a1/b1 also received MBON input onto both its dendrite and axon, and both AD1b2 and PD2a1/b1 receive axonal input from MBON-α'2 a memory-relevant MBON. These parallels suggest both a shared circuit motif and a relation between (innate) approach behaviour and memory (Dolan, 2019).

In general however, naive olfactory responses are more diverse than attraction or aversion, and olfactory stimulation can drive changes in locomotion/flight speed, stopping and turning or exploratory behaviour. This study identified one LH cell type which drives forward locomotion when stimulated, but does not impact valence behaviour. Much olfactory sensation occurs during flight and by recording the wingbeat responses of flying Drosophila before and after optogenetic stimulation it was possible to identify LH neurons that modulate different parameters (turning, wing thrust and wingbeat frequency) in flying animals. Interestingly, the effects of stimulating these neurons had different impacts on behaviour depending on visual stimulus (see multimodal integration below) while the persistence of these effects post-stimulation also varied. Therefore, different LH neurons likely drive different motor programs at various timescales. This diversity in downstream functions may explain why the LH has a large number of cell types which show distinct but frequently overlapping odour responses or pool different PN channels. Behavioural responses to odours (e.g. exploratory behaviour) may be a composite of different motor programs (e.g. locomotion increase, decrease turning) where each program is driven by a small assembly of different LH cell types. However, this study found only one LH neuron projecting to the sensorimotor integration circuitry of the central complex. Taken together these results imply that LH output is not immediately sent to motor neurons, likely it is integrated downstream with other information such as memory or internal state. This model is supported by the observation of specific impacts on motor behaviour by manipulations of different olfactory sensory neurons, although such manipulations also impact downstream MB circuits (Dolan, 2019).

Extensive overlap was found between a minority of LHONs and MB neurons, and an additional function of the LH may be to modulate and monitor the distributed coding in the MB. LHONs may modulate or even implant memories via DANs and the axoaxonic integration of MBON inputs by LHONs may occur more generally (Dolan, 2018). Higher olfactory neurons downstream of MB and LH likely read out olfactory information from these two different coding regimes to integrate decorrelated and hardwired olfactory representations (Dolan, 2019).

In addition to olfactory responses, the data also demonstrates the LH integrates multimodal input. This non-olfactory sensory information could provide context for the innate olfactory processing system which may be relevant for naive responses to odours or for pheremonally-driven behaviours such as courtship or aggression. Multimodal sensory input may also provide a route for direct integration of context and olfactory stimulation. Indeed, it was found the effects of optogenetic stimulation of LH neurons differed depending on the visual stimulation displayed to flying animals (Dolan, 2019).

In future experiments, the split-GAL4 tools described in this study will help investigators to determine the exact functions of the LH during olfaction at single cell type resolution. While the functions of individual LH neurons can range from aversion to memory retrieval, many LH neurons do not appear to be solely involved in avoidance or attraction. Higher-resolution behavioural assays will be needed. These will identify the subtle olfactory responses beyond avoidance or attraction and can be combined with connectomic information to design precise loss-of-function experiments (Dolan, 2019).

Both the mammalian and insect olfactory systems split into two parallel processing tracks, one hardwired or chemotropic and one distributed or random with functional roles in innate and learned behaviour respectively. This striking similarity suggests it may be able to obtain important and general insights into how olfactory perception is transformed into action using the simpler nervous system of Drosophila. Using the split-GAL4 lines generated in this study it was possible to provide a map of the inputs and outputs of this region, identifying many previously unknown features, such as multimodal input and extensive interactions with the MB. In addition this study has identified LH neurons that are sufficient to drive valence behaviour or specific motor movements, providing the first functional evidence that this hardwired region can direct diverse olfactory responses. This work represents a step towards a full model of how olfactory stimuli are processed in the Drosophila brain, from sensory input to motor output (Dolan, 2019).

Nutrient sensing by the central nervous system is emerging as an important regulator of systemic metabolism in both vertebrates and invertebrates. Little is known about how nutrition-dependent signals pass the blood brain barrier to convey this information. Like the vertebrate BBB, the BBB of Drosophila forms a tight barrier to passive transport, and is formed by highly conserved molecular components. Its simple structure and genetic accessibility make it an ideal model to study how nutritional signals are communicated to the CNS. Insulin and Insulin-like growth factors are conserved systemic signals that regulate growth and metabolism in response to nutrition. Although fruit flies do not have a single pancreas-like organ, they do produce eight distinct Drosophila Insulin/IGF-like peptides (Dilps) that are expressed in different tissues. A set of three Dilps (dILP2,3,5), released into circulation by Dilp-producing cells (IPCs) in the brain, have particularly important functions in regulating nutrition-dependent growth and sugar metabolism; ablation of IPCs in the CNS causes diabetes-like phenotypes, slows growth and development, and produces small, long-lived adult flies. Systemic Insulin/IGF signaling (IIS) increases in response to dietary sugars, proteins and lipids. Sugars act on IPCs directly to promote Dilp release, but other nutrients are sensed indirectly through signals from the fat body (an organ analogous to vertebrate liver/adipose tissue) (Brankatschk, 2014).

The Drosophila fat body produces two major types of lipoprotein particles: Lipophorin (LPP; Retinoid- and fatty acid-binding glycoprotein), the major hemolymph lipid carrier, and Lipid Transfer Particle (LTP). LTP transfers lipids from the intestine to LPP. These lipids include fatty acids from food, as well as from endogenous synthesis in the intestine. LTP also unloads LPP lipids to other cells (Van Heusden, 1989; Canavoso, 204; Parra-Peralbo, 2011). LPP crosses the BBB and accumulates throughout the brain. It is required for nutrition-dependent exit of neural stem cells from quiescence (Brankatschk, 2010). This study investigated possible functions of LTP in the brain (Brankatschk, 2014).

This work demonstrates a key requirement for lipoproteins in conveying nutritional information across the BBB to specific neurons in the brain. As particles that carry both endogenously synthesized and diet-derived lipids, lipoproteins are well-positioned to perform this function. The data suggest that transport of LTP across the BBB to Dilp2-recruiting neurons (DRNs) influences communication between DRNs and the Dilp-producing IPCs, increasing the release of Dilp2 into circulation. Since the IPCs also deliver Dilp2 to the DRNs, this indicates that these two neuronal populations may communicate bidirectionally. How might LTP affect the function of DRNs? One possibility is that it acts to deliver a signaling lipid to the DRNs. It could do so either directly, or indirectly by promoting lipid transfer from LPP, which is present throughout the brain. LTP enrichment on specific neurons may increase lipid transfer to these cells (Brankatschk, 2014).

This work highlights a key function for BBB cells in transmitting nutritional information to neurons within the brain. Feeding with yeast food increases free calcium in BBB glia, which then increases transport of LTP to DRNs. How might BBB cells detect the difference between yeast and plant food? The data suggest differences in the lipid composition of yeast and plant-derived foods are responsible. Previous work has shown that the lipids in these foods differ in their fatty acid composition. Yeast food has shorter and more saturated fatty acids than plant food (24). How could these nutritional lipids affect the activity of BBB glia? Interestingly, differences in food fatty acid composition are directly reflected in the fatty acids present in membrane lipids of all larval tissues including the brain. Thus, it is possible that the bulk membrane properties of BBB glia are different on these two diets. Membrane lipid composition is known to affect a variety of signaling events. Alternatively, yeast food may influence the specific fatty acids present in signaling lipids that activate BBB glia (Brankatschk, 2014).

This study demonstrates an unexpected functional specialization of the BBB glial network, which permits specific and regulated LTP transport to particular neurons. How this specificity arises is an important question for the future. It is noted that a subset of glial cells within the brain also accumulates LTP derived from the fat body. Could these represent specific transport routes from the BBB (Brankatschk, 2014)?

An alternative possibility is that transport depends on neuronal activity. Mammalian LRP1 promotes localized transfer of IGF in response to neuronal activity. Could LTP delivery by LRP1 and LRP2 (Megalin) in the Drosophila brain depend on similar mechanisms? The remarkable specificity of LTP trafficking in the Drosophila CNS provides a novel framework for understanding information flow between the circulation and the brain (Brankatschk, 2014).

To what extent might this be relevant to vertebrate systems? While it is clear that the vertebrate brain (unlike that of Drosophila) does not depend on lipoproteins to supply it with bulk sterols, this does not rule out possible functions for these particles in nutrient sensing. The vertebrate cerebrospinal fluid is rich in many types of HDL particles, including those containing ApoA-1, which is not expressed in the brain - this suggests that at least some lipoprotein particles in the brain may derive from the circulation. Consistent with this idea, ApoA-I can target albumin-containing nanoparticles across the BBB in rodents. Recent work suggests that lipoproteins may be the source of specific Long Chain Fatty Acids that signal to the hypothalamus to regulate glucose homeostasis, since neuronal lipoprotein lipase is required for this process. Thus, it would be interesting to investigate whether circulating mammalian lipoproteins might reach a subset of neurons in the hypothalamus (Brankatschk, 2014).

It has been known for some time that increasing the amount of yeast in the diet of lab grown Drosophila melanogaster increases the rate of development and adult fertility, but reduces lifespan. This study shows that flies have evolved specific mechanisms to increase systemic IIS in response to yeast, independently of the number of calories in the diet or its proportions of sugars proteins and fats. What pressures could have driven the evolution of such mechanisms? In the wild, Drosophila melanogaster feed on rotting plant material and their diets comprise both fungal and plant components. Drosophila disperse yeasts and transfer them to breeding sites during oviposition improving the nutritional resources available to developing larvae. Yeast that are able to induce more rapid development of the agents that disperse them may propagate more efficiently. On the other hand, it has been noted that Drosophila species that feed on ephemeral nutrient sources like yeasts or flowers have more rapid rates of development than other species. It may be that, even within a single species, the ability to adjust developmental rate to the presence of a short-lived resource is advantageous. Humans subsist on diets of both plant and animal materials that during most of evolution have differed in their availability. It would be interesting to investigate whether Insulin/IGF signaling in humans might respond to qualitative differences in the lipid composition of these nutritional components (Brankatschk, 2014).

The impressive consistency and fidelity in size of developing organisms reflects both the robustness of genetic programs and the developmental plasticity necessary to counteract the variations in size arising from genetic noise, erroneous morphogenesis, disease, or injury. To counterbalance growth abnormalities, systemic homeostatic mechanisms are implemented that delay the onset of the reproductive stage of adulthood until a correct size of the individual and its body parts has been reached. Indeed, most animals initiate a pubertal transition only once a critical size and body mass has been achieved and generally, in the absence of tissue damage or growth abnormalities. However, the mechanisms underlying such homeostatic regulation have yet to be fully defined (Vallejo, 2015).

Recently, the secreted peptide Dilp8, a member of the insulin/relaxin-like family has been identified as a factor mediating homeostatic control in Drosophila melanogaster. During the larval (growth) stage, the expression of dilp8 declines as maturation proceeds, whereas its expression is activated when growth is disturbed. Hence, fluctuating Dilp8 levels provides a reliable read-out of overall growth status (e.g., deficit) and of the time needed to complete growth and Dilp8 also orchestrates hormonal responses that stabilize body size. This includes inhibiting the production of the steroid hormone ecdysone by the prothoracic gland (PG) until the elements or organs affected are recomposed and also slowing down growth rates of undamaged tissues to ensure affected organs catch up with normal tissues in order to the adult flies reach a normal body size, maintain body proportions and symmetry. Accordingly, in the absence of dilp8, mutant flies are incapable of maintaining such strict control over their size, as reflected by the exaggerated variation in terms of overall proportionality and imperfect bilateral symmetry. However, the receptor that transduces Dilp8 signals and its site of action remained unknown (Vallejo, 2015).

Two models can be envisioned to establish such homeostatic regulation: a 'central' mechanism that dictates coordinated adjustments in both the duration and rate of growth, and an 'endocrine' mechanism that involves sensing and processing Dilp8 signals directly by hormone-producing cells. In Drosophila, several anatomically separate neural populations regulate growth and maturation time by impinging directly on the ring gland (comprising the PG and the juvenile hormone-producing corpus allatum, CA). Thus, the receptors that transduce the Dilp8 signals of growth status may act directly or communicate with neurons that produce the prothoracicotropic hormone (PTTH) and/or the neurons of the pars intercerebralis, including the insulin-producing cells (IPCs), that synthesize and release insulin-like peptides Dilp2, Dilp3 and Dilp5. Insect PTTH neurons, which are analogous to the gonadotropin-releasing hormone (GnRH) neurons in mammals, signal the commitment to sexual reproduction by stimulating the production of ecdysone in the PG in order to terminate growth. The IPCs in the pars intercerebralis, a functional equivalent of the mammalian hypothalamus, integrate nutritional signals and modulate tissue growth accordingly. Manipulation of IPCs by genetic ablation, starvation, or mutations in the single insulin receptor leads to the generation of animals with smaller size. Similarly manipulations of the PTTH neuropeptide and neurons result in variations in size of the adult flies, leading to larger or smaller than normal flies due to an extension or acceleration of the larval period and delayed pupariation. The insulin receptor also directly activates synthesis of the juvenile hormone (JH) in the CA, a hormone that promotes growth and the juvenile programs, and of ecdysone production in the PG, again augmenting the variation in normal adult size. These observations may explain how environmental and internal influences by operating through individual IPCs or PTTH neurons enable body size variation and plasticity in developmental timing that can be vital for survival in changing environments. However, the origin of developmental stability and invariant body size may require different or more complex neural mechanisms from those involved in adaptive size regulation (Vallejo, 2015).

By employing a candidate approach and biochemical assays, this study demonstrates that the orphan relaxin receptor Lgr3 acts as a Dilp8 receptor. This study identifies the neuronal population molecularly defined by the lgr3 enhancer fragment R19B09 (Jenett, 2012) and shows it is necessary and sufficient to mediate such homeostatic regulation. Using a cyclic AMP sensor as an indicator of Lgr3 receptor activation in vivo and tools for circuit mapping, it was determined that a pair of these Lgr3 neurons is highly sensitive to Dilp8. These neurons display extensive axonal arborizations and appear to connect with IPCs and PTTH neurons to form a brain circuit for homeostatic body size regulation. These data identify the insulin genes, dilp3 and dilp5, the JH, and ecdysone hormone as central in developmental size stability. Collectively, these findings unveil a homeostatic circuit that forms a framework for studying how the brain stabilize body size without constraining the adaptability of the system to reset body size in response to changing needs (Vallejo, 2015).

The data presented provide strong evidence that Dilp8 signals for organismal and organ homeostatic regulation of size are transduced via the orphan relaxin receptor Lgr3 and that activation of Lgr3 in molecularly defined neurons mediates the necessary hormonal adjustments for such homeostasis. Human insulin/relaxin-like peptides are transduced through four GPCRs, RXFP1 to 4. RXFP1 and 2 are characterized by large extracellular domains containing leucine-rich repeats similar to fly Lgr3 and Lgr4 receptors, and like Lgr3 (this study), their activation by their cognate ligand binding results in an increase in cAMP production. RXFP3 is distinctly different in structure from fly Lgr3 and its biochemical properties are also distinct, but RXPF3 is analogous to fly Lgr3 in the sense that it is found in highest abundance in the brain, suggesting important central functions for relaxin 3/RXFP3. However, a function in pubertal development and/or growth control for vertebrate relaxin receptors is presently unknown (Vallejo, 2015).

The neuronal populations that regulate body size and, in particularly, how their regulation generate variations in body size (plasticity) in response to internal and environmental cues such as nutrition have been intensely investigated. Less is known about how the brain stabilizes body size to ensure developing organisms reach the correct, genetically determined size. In particular, it remains unknown how limbs, and other bilaterally symmetric traits, grow to match precisely the size of the contralateral limb and maintain proportion with other parts even when they are faced with perturbations. Paired organs are controlled by an identical genetic program and grow in the same hormonal environment, and yet, small deviations in size can happen as result of developmental stress, genetic noise, or injury. Imperfections in symmetry thus reflect the inability of an individual to counterbalance variations and growth abnormalities (Vallejo, 2015).

This study shows that without lgr3, the brain is unable to detect growth disturbances and more importantly, it is not able to adjust the internal hormonal environment to allocate additional time during development to restore affected parts or catch-up on growth. Without lgr3, the brain also cannot slow down the growth rate to compensate for the extra time for growth so that unaffected and affected tissues can grow in a harmonious manner so as to sustain normal size, proportionality and symmetry. Using a cAMP sensor, this study has been able to define a pair of neurons that are highly sensitive to Dilp8 (Vallejo, 2015).

Communication in neuronal networks is essential to synchronize and perform efficiently. Notably, although most neurons have only one axon, Lgr3 responding neurons display extensive axonal arborizations reminiscent of hub neurons (Bonifazi, 2009). GRASP analyses show that Lgr3 neurons are broadly connected with the IPCs, and to a lesser extent with PTTH neurons, linking (Dilp8) inputs to the neuronal populations that regulate the key hormonal outputs that modulate larval and imaginal disc growth. Furthermore, the information flow from Lgr3 neurons to IPCs and to PTTH may explain how the brain matches growth with maturation in response to Dilp8. This brain circuit provides the basis for studying how the brain copes with genetic and environmental perturbations to stabilize body size, proportions and symmetry that is vital for the animal's survival (Vallejo, 2015).

Routine statistical analysis for differentially expressed genes, including a multiple-testing correction across all 16 data sets, did not reveal significant differences at a false discovery rate (FDR) of <0.05. Therefore CARMAweb was used to identify 146 mRNAs whose average signal was >7 in αβ neurons and that were also higher than in α'β' neurons by a factor of >2. Of the top 60 transcripts from this list, 29 were significantly different from α'β' signals and represent transposons. Alignment of the corresponding values from the γ and no-MB profiles showed a similarly significant bias in transposon expression over these samples. Retrotransposons were identified that transpose via a replicative mechanism involving an RNA intermediate and DNA elements that use nonreplicative excision and repair. Retrotransposons can be subdivided into long-terminal repeat (LTR) elements and long interspersed nuclear elements (LINEs). Rleven LTR elements (Tabor, mdg1, roo, qbert, gypsy, invader3, gypsy2, microcopia, 412, accord, and blood), 11 LINE-like elements (G6, RT1b, HeT-A, Ivk, Cr1a, F element, Doc2, baggins, R2, Doc3, and Doc), and four DNA elements (Bari1, pogo, Tc3, and transib3) were identified (Perrat, 2013).

Fourteen transposons, representing the most abundant in each class, were further analyzed. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of RNA from independently purified cell samples confirmed that transposon expression was significantly higher in αβ neurons than in other MB neurons. All transposons, other than R2, were also significantly higher in αβ neurons than in the rest of the brain. R2 is unique, because it exclusively inserts in the highly repeated 28S rRNA locus and heterochromatin (Perrat, 2013).

Transposition is ordinarily regulated by chromatin structure and posttranscriptional degradation of transposon mRNA guided by complementary RNAs. The small interfering RNA (siRNA) pathway has been implicated in somatic cell. In contrast, the Piwi-interacting RNA (piRNA) pathway has a more established role in the germline. The microarray analysis skewed attention toward piRNA because the expression level of the translocated Stellate locus, Stellate12D orphon (Ste12DOR) mRNA, was higher in αβ than in other MB neurons and the rest of the brain by a factor of >20. Stellate repeat transcripts are usually curtailed by piRNA, not siRNA. Stellate repeats encode a casein kinase II regulatory subunit, and piRNA mutant flies form Stellate protein crystals in testis. Immunostaining Stellate in the brain labeled puncta within αβ dendrites in the MB calyx, consistent with high Ste12DOR expression in wild-type αβ neurons (Perrat, 2013).

piRNAs are loaded into the Piwi clade argonaute proteins Piwi, Aubergine (Aub), and Argonaute 3 (Ago3). Piwi and Aub can amplify piRNA pools with Ago3. To investigate piRNA involvement in differential transposon expression, Piwi proteins and colocalized GFP were immunolocalized to assign signals to MB neuron type. Aub and Ago3 differentially labeled MB subdivisions in addition to structures throughout the brain, but no Piwi was detected. The ellipsoid body of the central complex stained strongly for Aub but not at all for Ago3, which suggests possible functional exclusivity of Piwi proteins in the brain (Perrat, 2013).

Differential Aub and Ago3 labeling was most evident within axon bundles in the peduncle and lobes, where MB neuron types are anatomically discrete. Aub protein colocalized with γ and α'β' neurons in the peduncle and lobes but was reduced in αβ neurons in both locations. Ago3 did not label MB lobes but colocalized with γ neurons in the peduncle. Ago3 labeled core αβ (αβc) neurons but did not label outer αβ neurons. Therefore, outer αβ neurons do not abundantly express Aub or Ago3, which implies that transposon suppression is relaxed. In contrast, γ neurons express Aub and Ago3, providing potential for piRNA amplification, and α'β' neurons express Aub. These patterns of Aub and Ago3 in the MB peduncle appear conserved in brains from D. erecta, D. sechellia, and the more distantly related D. pseudoobscura species (Perrat, 2013).

Loss of siRNA function elevates transposon expression in the head. These findings were replicated with ago2414 and dcr-2L811fsX mutant flies. In parallel, trans-heterozygous aub (aubHN2/aubQC42) and ago3 heads (ago3t2/ago3t3) and trans-heterozygous armitage heads (armi1/armi72.1) were used to test whether piRNA suppressed transposon expression. Levels of the 14 LTR, LINE-like, and TIR group transposons verified to be expressed in αβ neurons were assayed by qRT-PCR; of these 14 transposons, 13 were significantly elevated in siRNA-defective ago2 and dcr-2 mutants. The piRNA-defective aub, ago3, and armi mutants also exhibited significantly elevated levels of 9 of the 14 elements. Levels of the LTR elements gypsy, Tabor, and qbert; the LINE-like elements HeT-A, RT1b, and R2; and the TIR element pogo were higher in ago3 mutants. In addition, blood, Tabor, and R2 were elevated in aub mutants, and blood, gypsy, Tabor, invader3, qbert, HeT-A, and R2 were elevated in armi mutants. Therefore, the piRNA pathway contributes to transposon silencing in the brain, and low levels of Aub and Ago3 may permit expression in αβ neurons (Perrat, 2013).

To determine whether transposons are mobile, new insertions were mapped by deep sequencing of αβ DNA. αβ neurons were purified by FACS, as for transcriptome analysis, but isolated genomic DNA. Insertions were defined by paired-end reads in which one end mapped to the annotated genome and the other to the transposon sequence. To identify de novo transposition events in αβ neurons, the genomic position of transposons within the αβ sequence were compared to those located by sequencing DNA from genetically identical embryos. In addition, DNA was sequenced from the remainder of the brain tissue from the FACS separation of αβ neurons (Perrat, 2013).

These studies identified 3890 transposon insertions in embryo DNA that differed from the published Drosophila genome sequence. In comparison, αβ neuron DNA revealed 215 additional sites. The remaining brain tissue uncovered 200 new insertions, including 19 that were identical to those in αβ neurons. The sequencing depth for embryos was an order of magnitude greater than for neurons because embryo material could be collected more easily; hence, the αβ and other brain insertions are likely de novo. By randomly sampling reads to yield 1x genome coverage, 129 new transposon insertions were calculated per αβ neuron genome. Sequencing single neurons would reveal the exact cellular frequency and heterogeneity of transposition events (Perrat, 2013).

New αβ insertions occurred across all chromosomes, without obvious regional bias. In addition, insertions resulted from 49 different transposons representing LTR, LINE-like, TIR, and Foldback (FB) classes. They included 11 of the 29 transposons in the αβ transcriptome, and the number of insertions per class was consistent with their prevalence in the genome. Therefore, many transposons mobilize in αβ neurons (Perrat, 2013).

Of the 215 de novo αβ insertions, 108 mapped close to identified genes. Of these, 35 disrupted exons, 68 disrupted introns, and 5 fell in promoter regions (<1 kb from transcription start site). The remaining 107 insertions mapped to piRNA clusters or intergenic regions and were not assigned to a particular gene. A similar distribution was observed for the 200 new insertions in the rest of the brain. The reference fly genome has 258 transposon insertions in exonic regions, 11,110 insertions in intronic regions, 502 insertions in promoter regions, and 33,008 insertions in intergenic regions. Therefore, both groups of brain cells had a significantly larger fraction of insertions within exons, and fewer in intergenic regions, than the transposons that are annotated in the genome. To test whether such a distribution was unique to neurons, de novo insertions were analyzed in ovary DNA, again using embryo sequence as the comparison. New insertions in ovary DNA revealed a similar skew toward exons (Perrat, 2013).

In mammals, active L1 elements appear to disrupt neurally expressed genes. New αβ neuron insertions, but not those in other tissue, were significantly enriched in 12 Gene Ontology (GO) terms, all of which are related to neural functions. Moreover, promoter regions from 18 of 20 of the targeted genes drive expression in αβ neurons. Exonic insertions were found in gilgamesh, derailed, and mushroom body defect and intronic insertions in dunce and rutabaga, all of which have established roles in MB development and function. In addition, MB neurons are principally driven by cholinergic olfactory projection neurons and receive broad GABA-ergic inhibition and dopaminergic modulation through G protein–coupled receptors. Intronic insertions were in nicotinic Acetylcholine Receptor α 80B, G protein-coupled receptor kinase 1, and cyclic nucleotide gated channel-like and an exonic insertion in GABA-B-receptor subtype 1. Transposon-induced mosaicism could therefore alter integrative and plastic properties of individual MB αβ neurons (Perrat, 2013).

These data establish that transposon-mediated genomic heterogeneity is a feature of the fly brain and possibly other tissues. Together with prior work in rodents and humans, thede results suggest that genetic mosaicism may be a conserved characteristic of certain neurons. Work in mammals indicates that L1 expression occurs because the L1 promoter is released during neurogenesis. The data are consistent with such a model and also support the idea that transposons avoid posttranscriptional piRNA silencing in adult αβ neurons (Perrat, 2013).

A recent study described a role for piRNA in epigenetic control of memory-related gene expression in Aplysia neurons. It is therefore possible that MB neurons differentially use piRNA to control memory-relevant gene expression and that transposon mobilization is an associated cost. Because tansposon expression was found in αβ neurons of adult flies, it is conceivable that disruptive insertions accumulate throughout life, leading to neural decline and cognitive dysfunction. Alternatively, permitting transposition may confer unique properties across the 1000 neurons in the αβ ensemble and potentially produce behavioral variability between individual flies in the population (Perrat, 2013).

Deciphering the signals that provide positional information is a central issue in understanding how cell divisions are oriented. This study addressed this question in the highly proliferative NBs in the Drosophila larval brain, which maintain their division axis from one cell cycle to the next in part by using an apical microtubule network as a spatial cue to specify their apico-basal polarity axis and consequently the orientation of mitosis. Attempts were made to understand why NBs only partially fail to maintain their division axis upon loss of this intrinsic polarizing cues, and it was found out that the last-born daughter cell of NBs participates to their division axis maintenance. These results also shed light on some aspects of the physiological importance of division axis maintenance in larval NBs, which has remained elusive. Control of NB division orientation may provide a means to maximize NB/cortex glia surface area to allow optimum protection against environmental stresses by the cortex glia (Loyer, 2018).

Under normal conditions, about 80% of the surface of NBs is in direct contact with a cortex glia and NBs with partially defective division axis maintenance display reduced contact with cortex glia. This most likely directly results from NBs producing progeny between themselves and the cortex glia when the last-born daughter cell derived cue that positions normally the apico-basal polarity axis is damaged. This seems to be important for the protective function of these glial cells on NB proliferation under stress conditions. Indeed, NBs with reduced surface contact to cortex glia appear to be less well protected by glial cells, as was observed a significant increase of sensitivity to oxidative stress using an established assay. However, despite this reduction being statistically significant, only a 9% reduction was measured in NB/cortex glia contact area. On a normal diet, addition of the oxidant tert-butyl hydroperoxide (tbh) results in a 14% drop in NB proliferation when the formation of lipid droplets mediating this protection is prevented. It is therefore surprising that in these experiments reducing the NB/cortex glia contact area by only ~9% in Cindr-depleted NB is already accompanied by a similar drop in proliferation upon tbh treatment. Therefore, although this decrease may directly result from interfering with the protection provided by cortex glia, other unrelated functions of Cindr in protecting NBs against the effect of tbh cannot be ruled out (Loyer, 2018).

It was initially hypothesized that the last-born GMC could act as an additional, extrinsic cue maintaining NB division orientation. A number of observations are consistent with this possibility: it was observed that, upon (perhaps artefactual) last-born GMC movements, NBs realign their division axis toward this GMC; ablation of the last-born GMC and depletion of proteins specifically observed at the last-born GMC/NB interface affect division axis maintenance by misorienting the apico-basal polarity of NBs. It cannot be excluded that the entire NB and any intrinsic spatial cue that it carries simply rotate upon migration or ablation of the last-born GMC or depletion of proteins specifically observed at the last-born GMC/NB interface. Thus the last-born GMC may participate in division axis maintenance by preventing NB rotation. This function could be mediated by specific adhesive contacts at the interface with the NB, plausible given the numerous specific characteristics that were observed at that interface. In particular, the midbody carried by this interface, although not likely to act itself as a stable physical link given its possible ability to migrate within the fluid mosaic of the plasma membrane and the fact that its internalization does not affect division orientation maintenance, may be able to organize specific adhesive contacts at the NB/last-born GMC interface (Loyer, 2018).

An alternative hypothesis is that the last-born GMC provides a cue that more directly functions in specifying the orientation of the apico-basal polarity axis by polarizing Baz, which functions upstream of NB division orientation control. Consistently, despite affecting division orientation maintenance, neither GMC ablation nor RNAi of Cindr disrupt alignment of the mitotic spindle with the polarity axis. In this case, the molecular mechanism through which a positional information provided by the last-born GMC is transduced to the NB polarization machinery remains to be determined. Although bearing similarities with division axis maintenance in budding yeasts, relying on a Septin-rich cytokinesis remnant, the midbody of NBs is unlikely to directly control polarization as midbody internalization does not affect division axis maintenance. Instead, it is proposed that the midbody may organize various other specific components of the last-born GMC/NB interface that in turn may directly control NB polarization. This could be the case of cell-cell contacts organized by the midbody, consistent with the involvement of an adhesion molecule such as Roughest, whose mammalian orthologue physically interacts with Septins, and the fact that GMC ablation, although not directly targeting the interface, affects division axis maintenance. Another promising candidate potentially controlling NB polarity are the plasma membrane tubules probably organized by the midbody, given their physical origin (the midbody) and the timing (immediately after cytokinesis) of their appearance. Interestingly, a physical interaction was observed between Septins and the mammalian orthologue of Cindr, found enriched at the tubules and involved in division axis maintenance. Tubules function might be linked to the integrity of the last-born GMC/NB interface, which itself probably depends on the integrity of the last-born GMC. While these tubules do not disappear upon GMC ablation, it would be of particular interest to monitor whether tubules morphology, dynamics or the enrichment of Flare and Cindr are affected by ablation of the last-born daughter cell (Loyer, 2018).

Interestingly, proteins that were found to be involved in division axis maintenance were described to interact with polarity complexes in other contexts: Septins genetically interact with Baz during Drosophila embryogenesis, and the mammalian orthologues of Roughest regulate podocyte polarity by physically interacting with Par-3. However, both Septins and Roughest localize to the basal pole of NBs, whereas Baz polarizes apically. Therefore, how could a cue received at the basal pole direct polarization of Baz, at the opposite apical pole of the NB? In the C. elegans zygote, the sperm entry point acts as a cue inducing an actomyosin flow establishing Par complex polarity at the opposite end of the cell. Septins, Cindr, Roughest and Flare can be linked in one way or another to the regulation of actomyosin, and at least the maintenance of Baz localization in mitotic NBs is also actin-dependent. Intracellular long-range control of polarization has been further observed in eight-cell stage mouse blastomeres, where cell-cell contacts induce apical polarization at the opposite end of the cell. A promising lead for future work is the possible involvement of actomyosin-dependent mechanical forces in such long-range control of polarity in NBs. Indeed, tensions participate in polarization in the C. elegans zygote, were proposed to mediate polarization of eight-cell stage mouse blastomeres and maintain polarity in migrating neutrophils (Loyer, 2018).